HA binding agents

ABSTRACT

This disclosure relates to novel peptide agents, e.g., antibodies and antigen-binding fragments thereof, that bind hemagglutinin protein of influenza viruses, and methods of their use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/830,367, filed on Mar. 14, 2013, now granted as U.S. Pat. No.9,969,794, issued May 15, 2018, which claims priority to U.S.Application Ser. No. 61/645,554, filed on May 10, 2012, and U.S.Application Ser. No. 61/716,447, filed on Oct. 19, 2012. The disclosuresof the prior applications are considered part of (and are incorporatedby reference in) the disclosure of this application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 24, 2013, isnamed P2029-700110_SL.txt and is 186,454 bytes in size.

FIELD OF INVENTION

This disclosure relates to novel binding agents, e.g., peptide agents,e.g., antibody molecules, e.g., antibodies, and antigen-bindingfragments thereof, that bind hemagglutinin protein of influenza viruses,and in embodiments neutralize the virus, and methods of their use.

BACKGROUND

Influenza is an infectious disease caused by RNA viruses of the familyOrthomyxoviridae (the influenza viruses). Influenza viruses areclassified based on core protein into three genera A, B and C that arefurther divided into subtypes determined by the viral envelopeglycoproteins haemagglutinin (HA) and neuraminidase (NA). Influenza Aviruses infect a range of mammalian and avian species, whereas type Band C infections are largely restricted to humans. Only types A and Bcause human disease of any concern.

High mutation rates and frequent genetic reassortments of the influenzaviruses contribute to great variability of the HA and NA antigens. Minorpoint mutations causing small changes (“antigenic drift”) occurrelatively often. Antigenic drift enables the virus to evade immunerecognition, resulting in repeated influenza outbreaks duringinterpandemic years. Major changes in the HA antigen (“antigenic shift”)are caused by reassortment of genetic material from different influenzaA subtypes. Antigenic shifts resulting in new pandemic strains are rareevents, occurring through reassortment between animal and humansubtypes, for example in co-infected pigs.

Influenza A spreads around the world in seasonal epidemics, resulting inthe deaths of between 250,000 and 500,000 people every year, and up tomillions in some pandemic years. On average 41,400 people died each yearin the United States between 1979 and 2001 from influenza.

SUMMARY

The disclosure is based, at least in part, on the discovery of humananti-HA antibodies comprising functional and structural propertiesdisclosed herein, e.g., antibodies that bind a conserved region orepitope on influenza virus and uses thereof.

Accordingly, the disclosure features binding agents, e.g., antibodymolecules, or preparations, or isolated preparations thereof, that bindhemagglutinin (HA) from influenza viruses. In an embodiment, a bindingagent, e.g., an antibody molecule, is broad spectrum, and binds morethan one HA, e.g., an HA from one or both of Group 1 or Group 2 strainsof influenza A viruses and/or one or more strains of influenza Bviruses. Therefore, in some embodiments, a binding agent, e.g., anantibody molecule, featured in the disclosure can treat or preventinfection by a Group 1 influenza virus and a Group 2 influenza virus. Inother embodiments, a binding agent, e.g., an antibody molecule, featuredin the disclosure can treat or prevent infection by an influenza A virusand an influenza B virus. The binding agents, e.g, antibody molecules,share sufficient structural similarity with antibodies or variableregions disclosed herein such that they possess functional attributes ofthe antibodies disclosed herein. In embodiments the structuralsimilarity can be in terms of three dimensional structure or linearamino acid sequence or both.

In one aspect, the disclosure features an anti-hemagglutinin (anti-HA)binding agent, e.g., a specific binding agent, e.g., an antibodymolecule, or preparation, or isolated preparation thereof, comprisingone or more or all of the following properties:

(a) it fails to produce any escape mutants as determined by the failureof a viral titer to recover following at least 10, 9, 8, 7, 6, or 5rounds of serial infections in cell culture with a mixture of theantibody molecule and an influenza A virus, e.g., a Group 1 strain,e.g., an H1N1 strain, e.g., A/South Carolina/1/1918, A/PuertoRico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004;

(b) it produces fewer escape mutants than does a reference anti-HAantibody molecule, e.g., Ab 67-11, F16, F128, C179, F10, CR9114, orCR6261, e.g., when tested by the method described in (a);

(c) it prevents infection by at least 1, 2, 3, 4 or 5 influenza subtypesof Group 1, and by at least 1, 2, 3, 4 or 5 influenza subtypes of Group2;

(d) it inhibits fusogenic activity of the targeted HA;

(e) it treats or prevents infection by a Group 1 virus, such as wherethe virus is an H1, H5, or H9 virus; and it treats or prevents infectionby a Group 2 virus, such as where the virus is an H3 or H7 virus;

(f) it treats or prevents infection by influenza A strains H1N1 andH3N2;

(g) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with H1N1 and H3N2 when administered at 50 mg/kg, 25mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1mg/kg;

(h) it treats or prevents infection by influenza A H5N1 strains;

(i) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with H5N1 when administered at 50 mg/kg, 25 mg/kg, 10mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg;

(j) the concentration of antibody molecule required for 50%neutralization of influenza A virus is less than 10 μg/mL;

(k) it treats or prevents infection by an influenza B virus, e.g.,B/Wisconsin/1/2010;

(l) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with an influenza B virus, e.g., B/Wisconsin/1/2010,when administered at 10 mg/kg, 6 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1mg/kg;

(m) the concentration of antibody molecule required for 50%neutralization of influenza B virus, e.g., B/Wisconsin/1/2010, virus isless than 10 μg/mL;

(n) it prevents or minimizes secondary infection (e.g., secondarybacterial infection) or effects thereof on a subject;

(o) it is effective for preventing or minimizing secondary infection(e.g., secondary bacterial infection) or effects thereof on a subjectwhen administered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg;

(p) it binds an epitope which comprises or consists of the hemagglutinintrimer interface; and

(q) it binds an epitope other than that bound by a reference anti-HAantibody molecule, e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, orCR6261, e.g., as determined by structural analysis, e.g., by X-raycrystallography or NMR spectroscopy;

(r) in an embodiment it binds to an epitope, e.g., it has an epitopethat overlaps with or is the same as, of an antibody disclosed herein,e.g., as determined by mutational analysis or crystal structure analysis

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, has one or more of the following characteristics: the anti-HAantibody molecule prevents infection by at least 1, 2, 3, 4 or 5influenza subtypes of Group 1, and by at least 1, 2, 3, 4 or 5 influenzasubtypes of Group 2; the concentration of the anti-HA antibody moleculerequired for 50% neutralization of influenza A virus is less than 10μg/mL; or the anti-HA antibody molecule binds an epitope that comprisesor consists of the hemagglutinin trimer interface.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, featured in the disclosure treats or prevents infection by aGroup 1 virus, such as where the virus is an H1, H2, H5, H6, H8, H9,H12, H11, H13, H16, or H17 virus; and treats or prevents infection by aGroup 2 virus, such as where the virus is an H3, H4, H7, H10 or H15virus.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, featured in the disclosure prevents infection by at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 influenza subtypes of Group 1, and byat least 1, 2, 3, 4, 5 or 6 influenza subtypes of Group 2.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, featured in the disclosure treats or prevents infection by oneor more of H1N1, H2N2, H5N1, and H9N2, and also treats or preventsinfection by one or more of H3N2 and H7N7.

In an embodiment, a binding agent, e.g., antibody molecule, binds, andin embodiments, neutralizes:

at least one strain from the Group 1 H1, e.g., H1 a or H1b, cluster andat least one strain from the Group 2 H3 or H7 cluster.

In an embodiment, a binding agent, e.g., antibody molecule, binds, andin embodiments, neutralizes:

at least one strain from the Group 1 H1, e.g., H1 a or H1b, cluster andat least one influenza B strain, e.g., B/Wisconsin/1/2010.

In an embodiment, a binding agent, e.g., antibody molecule, binds, andin embodiments, neutralizes:

at least one strain from the Group 2 H3 or H7 cluster and at least oneinfluenza B strain, e.g., B/Wisconsin/1/2010.

In an embodiment, a binding agent, e.g., antibody molecule, binds, andin embodiments, neutralizes:

at least one strain from the Group 1 H1, e.g., H1 a or H1b, cluster, atleast one strain from the Group 2 H3 or H7 cluster, and at least oneinfluenza B strain, e.g., B/Wisconsin/1/2010.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, featured in the disclosure treats or prevents infection by oneor more of influenza B viruses, e.g., B/Wisconsin/1/2010.

In one embodiment, the anti-HA antibody molecule is not an anti-HAantibody molecule previously described in the art. For example, theanti-HA antibody molecule is other than one or more or all of Ab 67-11(U.S. Provisional Application No. 61/645,453,), FI6 (FI6, as usedherein, refers to any specifically disclosed FI6 sequence in U.S.Published Application No. 2010/0080813, US published application No.2011/0274702, WO2013/011347 or Corti et al., Science 333:850-856, 2011,published online Jul. 28, 2011; FIGS. 12A to 12C), FI28 (U.S. PublishedApplication No. 2010/0080813), C179 (Okun et al., J. Virol.67:2552-1558, 1993), F10 (Sui et al., Nat. Struct. Mol. Biol. 16:265,2009), CR9114 (Dreyfus et al., Science. 2012; 337(6100):1343-1348;published online Aug. 9, 2012), or CR6261 (Ekiert et al., Science324:246-251, 2009; published online Feb. 26, 2009).

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, neutralizes infection with H1N1 and H3N2 in vitro. In anotherembodiment, binding agent, e.g., an anti-HA antibody molecule,neutralizes infection with H1N1 and H3N2 in vivo.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, neutralizes infection with H5N1 in vitro. In anotherembodiment, binding agent, e.g., an anti-HA antibody molecule,neutralizes infection with H5N1 in vivo.

In one embodiment, the binding agent, e.g., an anti-HA antibodymolecule, neutralizes infection with an influenza B virus, e.g.,B/Wisconsin/1/2010, in vitro. In another embodiment, the binding agent,e.g., an anti-HA antibody molecule neutralizes infection with aninfluenza B virus, e.g., B/Wisconsin/1/2010, in vivo.

In another embodiment, the concentration of the binding agent, e.g., ananti-HA antibody molecule, required for 50% neutralization of influenzaA virus is 10 μg/mL or less, such as 9 μg/mL or less, 8 μg/mL or less, 7μg/mL or less, 6 μg/mL or less, or 5 μg/mL or less.

In another embodiment, the concentration of the binding agent, e.g., ananti-HA antibody molecule, required for 60% neutralization of influenzaA virus, 50% neutralization of influenza A virus, or 40% neutralizationof influenza A virus is 10 μg/mL or less, such as 9 μg/mL or less, 8μg/mL or less, 7 μg/mL or less, 6 μg/mL or less, or 5 μg/mL or less.

In yet another embodiment, the binding agent, e.g., an anti-HA antibodymolecule, is effective for prevention or treatment of infection, e.g.,in humans or mice, with H1N1 and H3N2, such as when administered at 50mg/kg, 25 mg/kg, 10 mg/kg, 6.0 mg/kg, 5.0 mg/kg, 4.0 mg/kg, 3.0 mg/kg,2.0 mg/kg, 1.0 mg/kg or less.

In still another embodiment, the binding agent, e.g., an the anti-HAantibody molecule, is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H5N1, such as when administeredat 50 mg/kg, 25 mg/kg, 10 mg/kg, 6.0 mg/kg, 5.0 mg/kg, 4.0 mg/kg, 3.0mg/kg, 2.0 mg/kg, 1.0 mg/kg or less.

In another embodiment, a binding agent, e.g., an anti-HA antibodymolecule, is effective for the treatment or prevention of a Group 1virus, where the Group 1 virus is H1, H5, or H9, and in anotherembodiment, the binding agent, e.g., an anti-HA antibody molecule, iseffective for the treatment or prevention of a Group 2 virus, where theGroup 2 virus is H3 or H7.

In another embodiment, the concentration of the binding agent, e.g., ananti-HA antibody molecule, required for 50% neutralization of influenzaB virus, e.g., B/Wisconsin/1/2010, is 10 μg/mL or less, such as 9 μg/mLor less, 8 μg/mL or less, 7 μg/mL or less, 6 μg/mL or less, or 5 μg/mLor less.

In another embodiment, the concentration of the binding agent, e.g., ananti-HA antibody molecule, required for 60% neutralization of influenzaB virus, e.g., B/Wisconsin/1/2010, 50% neutralization of influenza Bvirus, e.g., B/Wisconsin/1/2010, or 40% neutralization of influenza Bvirus, e.g., B/Wisconsin/1/2010, is 10 μg/mL or less, such as 9 μg/mL orless, 8 μg/mL or less, 7 μg/mL or less, 6 μg/mL or less, or 5 μg/mL orless.

In another embodiment, the binding agent, e.g., an anti-HA antibodymolecule, is a full length tetrameric antibody, a single chain antibody(scFv), a F(ab′)₂ fragment, a Fab fragment, or an Fd fragment. Inanother embodiment, the heavy chain of the antibody molecule is a γ1heavy chain, and in yet another embodiment, the light chain of theantibody molecule is a κ light chain or a λ light chain. In yet anotherembodiment, the anti-HA antibody molecule featured in the disclosure isan IgG1 antibody.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and S292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and S329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

a and aa;

b and bb;

c and cc;

d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In an embodiment the binding agent, e.g., a specific binding agent,e.g., an antibody molecule, comprises one or both of:

a heavy chain variable region comprising at least, or more than, 60, 65,70, 75, 80, 85, 87, 90, 95, 98 or 99 percent homology with a heavy chainvariable region from Table 3, Table 4A, Table 4B, FIG. 2, FIG. 13 orFIG. 17; and

a light chain variable region comprising at least, or more than, 60, 65,70, 75, 80, 85, 87, 90, 95, 98 or 99 percent homology with light chainvariable region from Table 3, Table 4A, Table 4B, FIG. 3, FIG. 14 orFIG. 17.

In an embodiment, the antibody molecule comprises a heavy chain variableregion 25 (SEQ ID NO:25), or a structurally or functionally relatedvariable heavy chain region as described herein.

In an embodiment, the antibody molecule comprises a light chain variableregion 52 (SEQ ID NO:52), 155 (SEQ ID NO:155), or 45 (SEQ ID NO:45), ora structurally or functionally related variable light chain region asdescribed herein.

In an embodiment, the antibody molecule comprises:

a heavy chain variable region 25 (SEQ ID NO:25), or a structurally orfunctionally related variable heavy chain region as described herein;and

a light chain variable region 52 (SEQ ID NO:52), 155 (SEQ ID NO:155), or45 (SEQ ID NO:45), or a structurally or functionally related variablelight chain region as described herein.

In an embodiment, the antibody molecule comprises a heavy chain variableregion comprising one, two, or all of CDR1, CDR2, and CDR3, from heavychain variable region 25 (SEQ ID NO:25), or a structurally orfunctionally related variable heavy chain region as described herein.

In an embodiment, the antibody molecule comprises a light chain variableregion comprising one, two, or all of CDR1, CDR2, and CDR3, from lightchain variable region 52 (SEQ ID NO:52), 155 (SEQ ID NO:155), or 45 (SEQID NO:45), or a structurally or functionally related sequence asdescribed herein.

In an embodiment, the antibody molecule comprises:

a heavy chain variable region comprising one, two, or all of CDR1, CDR2,and CDR3, from heavy chain variable region 25 (SEQ ID NO:25), or astructurally or functionally related variable heavy chain region asdescribed herein; and

a light chain variable region comprising one, two, or all of CDR1, CDR2,and CDR3, from light chain variable region 52 (SEQ ID NO:52), 155 (SEQID NO:155), or 45 (SEQ ID NO:45), or a structurally or functionallyrelated variable light chain region as described herein.

In an embodiment the antibody molecule comprises a heavy chain variableregion from FIG. 2 or FIG. 13 or a structurally or functionally relatedvariable heavy chain region as described herein.

In an embodiment the antibody molecule comprises a light chain variableregion from FIG. 3 or FIG. 14 or a structurally or functionally relatedvariable light chain region as described herein.

In an embodiment the antibody molecule comprises one, two, or all of, aCDR1, CDR2, and CDR3 from a heavy chain variable region from FIG. 2 orFIG. 13, or a structurally or functionally related sequences asdescribed herein.

In an embodiment the antibody molecule comprises one, two, or all of, aCDR1, CDR2, and CDR3 from a light chain variable region from FIG. 3 orFIG. 14, or a structurally or functionally related sequences asdescribed herein.

In an embodiment the antibody molecule comprises one, two or all of, HCCDR1, HC CDR2, and HC CDR3 and one, two or all of, LC CDR1, LC CDR2, andLC CDR3 from an antibody disclosed in Table 3, or a structurally orfunctionally related sequences as described herein.

In another embodiment, the antibody molecule comprises the light chainLC45 (SEQ ID NO: 45). In yet another embodiment, the antibody comprisesthe light chain LC45, and the heavy chain HC25 (SEQ ID NO: 25) or 24(SEQ ID NO: 24). In one embodiment, the antibody molecule comprises thelight chain Ab032(SEQ ID NO: 45) and the heavy chain 25 (SEQ ID NO: 25).In yet another embodiment, the antibody molecule comprises light chainLC52(SEQ ID NO: 52) and heavy chain HC25 (SEQ ID NO: 25).

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from heavy chain disclosedherein. E.g., the antibody molecule comprises one or more or all of FR1,FR2, FR3, or FR4, or FR sequences that differ individually, orcollectively, by no more than 1, 2, 3, 4, of 5 amino acid residues,e.g., conservative residues, from a heavy chain disclosed herein; and

b) one or more framework regions (FRs) from light chain disclosedherein. E.g., the antibody molecule comprises one or more or all of FR1,FR2, FR3, or FR4, or FR sequences that differ individually, orcollectively, by no more than 1, 2, 3, 4, of 5 amino acid residues,e.g., conservative residues, from light chain disclosed herein.

In one aspect, an anti-HA antibody molecule featured in the disclosure,or preparation, or isolated preparation thereof, comprises

(a) a heavy chain immunoglobulin variable domain comprising a sequenceat least 60, 70, 80, 85, 87, 90, 95, 97, 98, or 99, e.g., 90%,homologous, to a heavy chain consensus sequence provided herein, e.g.,the heavy chain consensus sequence provided in FIG. 2 or FIG. 13, e.g.,the heavy chain consensus sequence provided in FIG. 2, SEQ ID NO:161;and

(b) a light chain immunoglobulin variable domain comprising a sequenceat least 60, 70, 80, 85, 87, 90, 95, 97, 98, or 99, e.g., 95%,homologous, to a light chain consensus sequence provided herein, e.g.,the light chain consensus sequence provided in FIG. 3 or FIG. 14, e.g.,the light chain consensus sequence provided in FIG. 3, SEQ ID NO:62.

For example, in one embodiment, the anti-HA antibody molecule featuredin the disclosure comprises one or both of:

(a) a heavy chain immunoglobulin variable domain comprising the sequenceof SEQ ID NO:161, or a sequence at least 87% identical to SEQ ID NO:161;and

(b) a light chain immunoglobulin variable domain comprising the sequenceSEQ ID NO:62, or a sequence at least 95% identical to SEQ ID NO:62.

In another embodiment the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable domain comprising the sequenceof SEQ ID NO:161, or a sequence at least 87% identical to SEQ ID NO:161;and

(b) a light chain immunoglobulin variable domain comprising the sequenceSEQ ID NO:62, or a sequence at least 95% identical to SEQ ID NO:62,wherein said antibody molecule:

(i) fails to produce any escape mutants as determined by the failure ofa viral titer to recover following at least 10, 9, 8, 7, 6, or 5 roundsof serial infections in cell culture with a mixture of the antibodymolecule and an influenza virus (e.g., an influenza A virus, e.g., aGroup 1 strain, e.g., an H1N1 strain, e.g., A/South Carolina/1/1918,A/Puerto Rico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., B/Wisconsin/1/2010); and

(ii) produces fewer escape mutants than does a reference anti-HAantibody molecule, e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, orCR6261, such as when tested by the method described in (i).

In an embodiment, the disclosure features an antibody moleculecomprising one or both of:

(a) a heavy chain immunoglobulin variable region comprising the sequenceof SEQ ID NO:161, or a sequence that differs from SEQ ID NO:161 by notmore than 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15 or 16, e.g., by nomore than 2, 3, 4, or 5 amino acids, e.g., conservative amino acids; and

(b) a light chain immunoglobulin variable domain comprising the sequenceSEQ ID NO:62, or a sequence that differs from SEQ ID NO:62 that differsby no more than 1, 2, 3, 4 or 5 amino acids, e.g., conservative aminoacids.

In one embodiment, the 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15 or 16amino acid differences, e.g., conservative amino acid differences, inthe heavy chain immunoglobulin variable region are in the FR regions ofthe heavy chain immunoglobulin variable domain. In another embodiment,the 1, 2, 3, 4 or 5 amino acid differences, e.g., conservative aminoacid differences, in the light chain immunoglobulin variable domain arein the FR regions of the light chain immunoglobulin variable domain. Inone embodiment, the amino acid differences in the heavy chainimmunoglobulin variable region, or in the light chain immunoglobulinvariable region, are conservative amino acid changes.

In an embodiment the binding agent, e.g., an antibody molecule, binds toan epitope, e.g., it has an epitope that overlaps with or is the sameas, of an antibody disclosed herein, e.g., as determined by mutationalanalysis or crystal structure analysis.

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from heavy chain consensussequence disclosed herein. e.g., the antibody molecule comprises one ormore or all of FR1, FR2, FR3, or FR4, or sequences that differindividually, or collectively, by no more than 1, 2, 3, 4, of 5 aminoacid residues, e.g., conservative residues, from heavy chain consensussequence disclosed herein; and

b) one or more framework regions (FRs) from light chain consensussequence disclosed herein. e.g., the antibody molecule comprises one ormore or all of FR1, FR2, FR3, or FR4, or sequences that differindividually, or collectively, by no more than 1, 2, 3, 4, of 5 aminoacid residues, e.g., conservative residues, from light chain consensusdisclosed herein.

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In another aspect, the disclosure features, a binding agent, e.g., anantibody molecule, or preparation, or isolated preparation thereof,comprising a structural or functional property of Ab 044.

In an embodiment, the antibody molecule competes with a referenceantibody molecule, e.g., an antibody molecule described herein, forbinding to a substrate, e.g., an HA. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-D-Y-K-N-Y-L-A                (SEQ ID NO:145);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:52; or    -   c) Ab 044.

The HA can be from a Group 1 strain, e.g., an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004.Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecules or the reference antibody molecule to decreasebinding of the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g.from an H1N1 strain, e.g., A/South Carolina/1/1918, A/PuertoRico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004. Reduction of the ability tobind can be evaluated by methods in the art. Reduction of the ability tobind can be evaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, which the reference antibody molecule binds. In anembodiment the antibody molecule does not bind to the same epitope, or aportion thereof, which the reference antibody molecule binds.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, on HA, as does a reference antibody molecule, e.g. anantibody molecule disclosed herein. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-D-Y-K-N-Y-L-A                (SEQ ID NO:145);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:52; or    -   c) Ab 044.

The HA can be HA1 or HA5, e.g., from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004Binding tothe same epitope, or a portion thereof, can be shown by one or more of:

-   -   a) mutational analysis, e.g., binding to HA, or binding affinity        for HA, is decreased or abolished if a residue is mutated;    -   b) analysis, e.g., comparison, of the crystal structure of the        antibody molecule and HA and the crystal structure of a        reference antibody and HA, e.g., to determine the touch points        of each;    -   c) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, from, e.g., an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004; and    -   d) (c) and one or both of (a) and (b).

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, from, e.g., anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004.

Reduction of the ability to bind can be evaluated by methods in the art.Reduction of the ability to bind can be evaluated, e.g., by one or moreof:

-   -   a) BIAcore analysis;    -   b) ELISA assay; or    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 52.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 52,

wherein, each HC CDR differs by no more than 1, 2, 3, 4 or 5 aminoacids, e.g., 1 or 2, e.g., conservative amino acids, from thecorresponding CDR of SEQ ID NO: 25 and each LC CDR differs by no morethan 1, 2, 3, 4 or 5 amino acids, e.g., 1 or 2, e.g., conservative aminoacids, from the corresponding CDR of SEQ ID NO: 52.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 52,

wherein the antibody molecule comprises 1, 2, 3, 4, 5, or all of:

-   -   (i) a HC CDR1 comprising: S at the 1st position and A at the 3rd        position in HC CDR1;    -   (ii) a HC CDR2 comprising one or both, e.g., one of: V at the        2^(nd) position; or N at the 7^(th) position and Q at the 16th        position in HC CDR2;    -   (iii) a HC CDR3 comprising: R at the 3rd position (and        optionally, L at the 3^(rd) position);    -   (iv) a LC CDR1 comprising one or both of, e.g., one of: I at the        3rd position; or D at the 6th position in LC CDR1;    -   (v) a LC CDR2 comprising one, two, or three of, e.g., one of: G        at the 2^(nd) position; Y at the 4^(th) position; or L at the        5^(th) position in LC CDR2;    -   (vi) a LC CDR3 comprising: S at the 9^(th) position in LC CDR3;

In an embodiment, the binding agent, e.g., an antibody molecule,comprises:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO:25 (or a sequence that differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., conservative amino acids, therefrom); and

(b) a light chain variable region segment comprising SEQ ID NO:52 (or asequence that differs by no more than 1, 2, 3, 4 or 5 amino acids, e.g.,conservative amino acids, therefrom).

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);        and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence:        -   Q-S-I-T-F-D-Y-K-N-Y-L-A (SEQ ID NO:145) (or a sequence that            differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2            amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom).

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a) LC CDR1-3, that collectively, differ from the AB 044 LC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids; and

b) HC CDR1-3, that collectively, differ from the AB 044 HC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids.

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO: 25; and (b) a light chain variable region segment comprising SEQID NO:52.

In an embodiment, the binding agent is an antibody molecule comprisingone or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, or 3, e.g., 1 or 2,        amino acids, e.g., conservative amino acids, there from,        optionally provided that at least 1 or 2 of the highlighted        residue are not changed, e.g., both S and A are not changed);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom, optionally provided that at least 1, 2,        or 3 of the highlighted residues are not changed, e.g., V or        both N and Q or all three of V, N, and Q are not changed);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom,        optionally provided that R is not changed); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence:        -   Q-S-I-T-F-D-Y-K-N-Y-L-A (SEQ ID NO:145) (or a sequence that            differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2,            amino acids, e.g., conservative amino acids, therefrom,            optionally provided that at least for 2 of the highlighted            residues are not changed, e.g., I or D is not changed);        -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72)            (or a sequence that differs by no more than, 1, 2, 3, 4, or            5, e.g., 1 or 2, amino acids, e.g., conservative amino            acids, therefrom, optionally provided that at least 1, 2 or            3 of the highlighted residues are not changed, e.g., 1, 2 or            all of G, Y, and L are not changed);        -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S (SEQ ID            NO:73) (or a sequence that differs by no more than, 1, 2, 3,            4, or 5, e.g., 1 or 2, amino acids, e.g., conservative amino            acids, therefrom, optionally provided that at least 1 or            both of the highlighted residues are not changed, e.g., S is            not changed).

In an embodiment a CDR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that CDR, (i.e., while other residues in that CDR might bechanged, the highlighted residue or combination of residues, are notchanged). E.g., in an embodiment, V or both N and Q, for heavy chainCDR2 are not changed.

In an embodiment a CDR of the light and a CDR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In an embodiment each of two CDRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of the three CDRs in the heavy chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the three CDRs in the light chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the six CDRs in the heavy and light chainincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In one embodiment, the binding agent is an antibody molecule thatcomprises one or more or all of the following properties:

(a) both S and A in HC CDR1 are unchanged;

(b) V or both N and Q or all three of V, N, and Q in HC CDR2 areunchanged;

(c) R in HC CDR3 is unchanged;

(d) One or both of I and D in LC CDR1 are unchanged.

(e) 1, 2 or 3 of G, Y and L in LC CDR2 are unchanged; OR

(f) S in LC CDR3 is unchanged.

In an embodiment the antibody molecule comprises 1, 2, 3, 4, 5, or all 6properties selected from (a) to (f).

In an embodiment, the antibody molecule comprises a heavy chain having aone or more properties selected from (a), (b), and (c) and a light chainhaving one or more properties selected from (d), (e), and (f).

In one embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising:

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence Q-S-I-T-F-D-Y-K-N-Y-L-A (SEQ ID        NO:145);

a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72); and

a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73).

In some embodiments, the antibody molecule comprises one or more or allof the following properties: (i) it fails to produce any escape mutantsas determined by the failure of a viral titer to recover following atleast 10, 9, 8, 7, 6, or 5 rounds of serial infections in cell culturewith a mixture of the antibody molecule and an influenza virus (e.g., aninfluenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g.,A/South Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,or an H5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, oran influenza B virus, e.g., B/Wisconsin/1/2010); and (ii) it producesfewer escape mutants than does a reference anti-HA antibody molecule,such as Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, such as whentested by the method described in (i).

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from SEQ ID NO: 25 e.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 25; and

b) one or more framework regions (FRs) from SEQ ID NO: 52. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 52.

In one embodiment, the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable region segment that furthercomprises one or more or all of:

an FR1 comprising the sequenceQ-V-Q-L-L-E-T-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ IDNO:74) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that T is not changed);

an FR2 comprising the sequence W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ IDNO:75) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that W is not changed, or that if changed, is otherthan R);

an FR3 comprising the sequenceR-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K (SEQ IDNO:76) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that one, two or three of I, R, or L is not changed,or that if I is changed it is other than G, if R is changed it is otherthan P. or if L is changed it is other than A); and

an FR4 comprising the sequence W-G-Q-G-T-T-L-T-V-S-S (SEQ ID NO:77) (ora sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2,amino acids, e.g., conservative amino acids, therefrom) orW-G-Q-G-T-T-V-T-V-S-S(SEQ ID NO:171) (or a sequence that differs by nomore than, 1, 2, 3, 4, or 5, e.g., 1 or 2, amino acids, e.g.,conservative amino acids, therefrom); and

(b) a light chain immunoglobulin variable region segment comprising oneor more or all of:

an FR1 comprising the sequenceD-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C-R-S-S(SEQ ID NO:78) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2,amino acids, e.g., conservative amino acids, therefrom, optionallyprovided that R is not changed);

an FR2 comprising the sequence W-Y-Q-Q-K-P-G-K-A-P-K-L-L-I-Y (SEQ IDNO:79) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom);

an FR3 comprising the sequenceG-V-P-S-R-F-S-G-S-G-S-G-T-D-F-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C (SEQ IDNO:80) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that C is not changed, or if changed, is other thanP); and

an FR4 comprising the sequence F-G-Q-G-T-K-V-E-I-K (SEQ ID NO:81) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom).

In an embodiment a FR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that FR, (i.e., while other residues in that FR might bechanged, the highlighted residue or combination of residues, are notchanged). E.g., in an embodiment, one, two or three of I, R, or L forheavy chain FR3 is not changed.

In an embodiment a FR of the light and a FR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that FR.

In an embodiment each of two FRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that FR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of FR2 and FR3 in the heavy chain includes one ofthe highlighted residues, or one of the highlighted combinations ofresidues, for that FR.

In an embodiment each of FR1 and FR2 in the heavy and light chainincludes one of the highlighted residues for that FR.

In an embodiment all of the highlighted residues in heavy chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in light chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in both heavy and lightchain FR1-4 are unchanged.

In an embodiment, sequence of FR1 of the heavy chain variable regionsegment is Q-V-Q-L-L-E-T-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T(SEQ ID NO:74).

In an embodiment, sequence of FR1 of the heavy chain variable regionsegment is E-V-Q-L-L-E-S-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T(SEQ ID NO:183).

In another embodiment, the binding agent, e.g., an antibody molecule,comprises one or more or all of the following properties: (a) it failsto produce any escape mutants as determined by the failure of a viraltiter to recover following at least 10, 9, 8, 7, 6, or 5 rounds ofserial infections in cell culture with a mixture of the antibodymolecule and an influenza virus (e.g., an influenza A virus, e.g., aGroup 1 strain, e.g., an H1N1 strain, e.g., A/South Carolina/1/1918,A/Puerto Rico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., B/Wisconsin/1/2010); (b) it produces fewer escape mutants thandoes a reference anti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28,C179, or CR6261, e.g., when tested by the method described in (a); (c)it binds with high affinity to a hemagglutinin (HA) of at least 1, 2, 3,4 or 5 influenza subtypes of Group 1 and at least 1, 2, 3, 4 or 5influenza subtypes of Group 2; (d) it treats or prevents infection by atleast 1, 2, 3, 4 or 5 influenza subtypes of Group 1, and by at least 1,2, 3, 4 or 5 influenza subtypes of Group 2; (e) it inhibits fusogenicactivity of the targeted HA; (f) it treats or prevents infection by aGroup 1 virus, wherein the virus is an H1, H5, or H9 virus; and treatsor prevents infection by a Group 2 virus, wherein the virus is an H3 orH7 virus; (g) it treats or prevents infection by influenza A strainsH1N1 and H3N2; (h) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H1N1 and H3N2 when administeredat 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2mg/kg or 1 mg/kg; (i) it treats or prevents infection by influenza Astrains H5N1; (j) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H5N1 when administered at 50mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kgor 1 mg/kg; (k) it binds with high affinity to a hemagglutinin (HA) ofan influenza B virus, e.g., B/Wisconsin/1/2010; (1) it treats orprevents infection by an influenza B virus, e.g., B/Wisconsin/1/2010;(m) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with an influenza B virus, e.g., B/Wisconsin/1/2010 whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (n) the concentration of antibody moleculerequired for 50% neutralization of influenza A virus is less than 10μg/mL; (o) the concentration of antibody molecule required for 50%neutralization of influenza B virus, e.g., B/Wisconsin/1/2010, is lessthan 10 μg/mL; (p) it prevents or minimizes secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject; (q) itis effective for preventing or minimizing secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (r) it binds an epitope which comprises orconsists of the hemagglutinin trimer interface; and (s) it binds anepitope other than that bound by a reference anti-HA antibody molecule,e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, e.g., whentested by a method disclosed herein, e.g., by competition in an ELISAassay.

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and S292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and S329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

-   -   a and aa;    -   b and bb;    -   c and cc;    -   d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In another aspect, the disclosure features, a binding agent, e.g., anantibody molecule, or preparation, or isolated preparation thereof,comprising a structural or functional property of Ab 069.

In an embodiment, the antibody molecule competes with a referenceantibody molecule, e.g., an antibody molecule described herein, forbinding to a substrate, e.g., an HA. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-E-Y-K-N-Y-L-A                (SEQ ID NO:172);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:155; or    -   c) Ab 069.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004.Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g. from anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay;    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more. In anembodiment the antibody molecule binds to the same epitope, or a portionthereof, which the reference antibody molecule binds. In an embodimentthe antibody molecule does not bind to the same epitope, or a portionthereof, which the reference antibody molecule binds.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, on HA, as does a reference antibody molecule, e.g. anantibody molecule disclosed herein. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-E-Y-K-N-Y-L-A                (SEQ ID NO:172);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:155; or    -   c) Ab 069.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004. Binding tothe same epitope, or a portion thereof, can be shown by one or more of:

-   -   a) mutational analysis, e.g., binding or lack thereof to mutant        HA, e.g., if a residue is mutated;    -   b) analysis, e.g., comparison, of the crystal structure of the        antibody molecule and HA and the crystal structure of a        reference antibody and HA, e.g., to determine the touch points        of each;    -   c) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, from, e.g., an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004; or    -   d) (c) and one or both of (a) and (b);

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g. from anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay;    -   c) flow cytometry. The antibody molecule can compete with the        reference antibody such that binding of the reference antibody        is decreased by 50% or more.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 155.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 155,

wherein each HC CDR differs by no more than 1, 2, 3, 4 or 5 amino acids,e.g., 1 or 2, e.g., conservative amino acids, from the corresponding CDRof SEQ ID NO: 25 and each LC CDR differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., 1 or 2, e.g., conservative amino acids, from thecorresponding CDR of SEQ ID NO: 155.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising at least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising at least 60, 70, 80, 85,90, 95, 98 or 99 percent homology with SEQ ID NO: 155,

wherein the antibody molecule comprises 1, 2, 3, 4, 5, or all of:

-   -   (i) a HC CDR1 comprising: S at the 1st position and A at the 3rd        position in HC CDR1;    -   (ii) a HC CDR2 comprising one or both, e.g., one of: V at the        2nd position; or N at the 7^(th) position and Q at the 16^(th)        position in HC CDR2;    -   (iii) a HC CDR3 comprising: R at the 3rd position (and        optionally, L at the 3^(rd) position);    -   (iv) a LC CDR1 comprising one or both of, e.g., one of: I at the        3rd position; or E at the 6th position in LC CDR1;    -   (v) a LC CDR2 comprising one, two or three of, e.g., one of: G        at the 2nd position; Y at the 4^(th) position; or L at the        5^(th) position in LC CDR2;        -   (vi) a LC CDR3 comprising: S at the 9^(th) position in LC            CDR3;

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO:25 (or a sequence that differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., conservative amino acids, therefrom); and

(b) a light chain variable region segment comprising SEQ ID NO:155 (or asequence that differs by no more than 1, 2, 3, 4 or 5 amino acids, e.g.,conservative amino acids, therefrom).

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO: 25; and

(b) a light chain variable region segment comprising SEQ ID NO:155.

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);        and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence:        -   Q-S-I-T-F-E-Y-K-N-Y-L-A (SEQ ID NO: 172) or a sequence that            differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2            amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom).

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a) LC CDR1-3, that collectively, differ from the AB 069 LC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids; and

b) HC CDR1-3, that collectively, differ from the AB 069 HC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids.

In an embodiment, the binding agent is an antibody molecule comprisingone or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, or 3, e.g., 1 or 2        amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1 or 2 of the highlighted        residues are not changed, e.g., both S and A are not changed);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom, optionally provided that at least 1, 2,        or 3 of the highlighted residues are not changed, e.g., V or        both N and Q or all three of V, N, and Q are not changed);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom        optionally provided that, R is not changed); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence:        -   Q-S-I-T-F-E-Y-K-N-Y-L-A (SEQ ID NO: 172) or a sequence that            differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2            amino acids, e.g., conservative amino acids, therefrom,            optionally provided that at least 1 or 2 of the highlighted            residues are not changed, e.g., I or E is not changed);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S (SEQ ID NO:72) (or        a sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g.,        1 or 2 amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1, 2, or 3 of the highlighted        residues are not changed, e.g., 1, 2 or all of G, Y, and L are        not changed);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S (SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom, optionally provided that, at least one or both of the        highlighted residues are not changed, e.g., S is not changed).

In an embodiment a CDR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that CDR, (i.e., while other residues in that CDR might bechanged, the highlighted residue or combination of residues, are notchanged).

In an embodiment a CDR of the light and a CDR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In an embodiment each of two CDRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of the three CDRs in the heavy chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the three CDRs in the light chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the six CDRs in the heavy and light chainincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In one embodiment, the binding agent is an antibody molecule thatcomprises one or more or all of the following properties:

(a) both S and A in HC CDR1 are unchanged.

(b) V or both N and Q or all three of V, N, and Q in HC CDR2 areunchanged.

(c) R in HC CDR3 is unchanged.

(d) one or both of I and E in LC CDR1 are unchanged.

(e) 1, 2 or 3 of G, Y and L in LC CDR2 are unchanged;

(f) S in LC CDR3 is unchanged.

In an embodiment the antibody molecule comprises 1, 2, 3, 4, 5, or all 6properties selected from (a) to (f).

In an embodiment, the antibody molecule comprises a heavy chain having aone or more properties selected from (a), (b), and (c) and a light chainhaving one or more properties selected from (d), (e), and (f).

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising:

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence Q-S-I-T-F-E-Y-K-N-Y-L-A (SEQ ID        NO:172);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72); and    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73).

In some embodiments, the antibody molecule comprises one or more or allof the following properties: (i) it fails to produce any escape mutantsas determined by the failure of a viral titer to recover following atleast 10, 9, 8, 7, 6, or 5 rounds of serial infections in cell culturewith a mixture of the antibody molecule and an influenza virus (e.g., aninfluenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g.,A/South Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,or an H5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, oran influenza B virus, e.g., B/Wisconsin/1/2010); and (ii) it producesfewer escape mutants than does a reference anti-HA antibody molecule,such as Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, such as whentested by the method described in (i).

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from SEQ ID NO: 25. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 25; and

b) one or more framework regions (FRs) from SEQ ID NO: 155. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 155.

In one embodiment, the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable region segment that furthercomprises one or more or all of:

an FR1 comprising the sequenceQ-V-Q-L-L-E-T-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ IDNO:74) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that T is not changed);

an FR2 comprising the sequence W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ IDNO:75) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that W is not changed, or that if changed, is otherthan R);

an FR3 comprising the sequenceR-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K (SEQ IDNO:76) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that one, two or three of I, R, or L is not changed,or that if I is changed it is other than G, if R is changed it is otherthan P. or if L is changed it is other than A); and

(b) the light chain immunoglobulin variable region segment comprises oneor more or all of

an FR1 comprising the sequenceD-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C-R-S-S(SEQ ID NO:78) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom, optionallyprovided that R is not changed);

an FR2 comprising the sequence W-Y-Q-Q-K-P-G-K-A-P-K-L-L-I-Y (SEQ IDNO:79) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom);

an FR3 comprising the sequenceG-V-P-S-R-F-S-G-S-G-S-G-T-D-F-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C (SEQ IDNO:80) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that C is not changed, or if changed, is other thanP); and

an FR4 comprising the sequence F-G-Q-G-T-K-V-E-I-K (SEQ ID NO:81) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom).

In an embodiment a FR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that FR, (i.e., while other residues in that FR might bechanged, the highlighted residue or combination of residues, are notchanged). E.g., in an embodiment, one, two or three of I, R, or L forheavy chain FR3 is not changed.

In an embodiment a FR of the light and a FR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that FR.

In an embodiment each of two FRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that FR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of FR2 and FR3 in the heavy chain includes one ofthe highlighted residues, or one of the highlighted combinations ofresidues, for that FR.

In an embodiment each of FR1 and FR2 in the heavy and light chainincludes one of the highlighted residues for that FR.

In an embodiment all of the highlighted residues in heavy chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in light chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in both heavy and lightchain FR1-4 are unchanged.

In another embodiment, the binding agent, e.g., an antibody molecule,comprises one or more or all of the following properties: (a) it failsto produce any escape mutants as determined by the failure of a viraltiter to recover following at least 10, 9, 8, 7, 6, or 5 rounds ofserial infections in cell culture with a mixture of the antibodymolecule and an influenza virus (e.g., an influenza A virus, e.g., aGroup 1 strain, e.g., an H1N1 strain, e.g., A/South Carolina/1/1918,A/Puerto Rico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., B/Wisconsin/1/2010); (b) it produces fewer escape mutants thandoes a reference anti-HA antibody molecule, e.g., Ab 67-11, F16, F128,C179, or CR6261, e.g., when tested by the method described in (a); (c)it binds with high affinity to a hemagglutinin (HA) of at least 1, 2, 3,4 or 5 influenza subtypes of Group 1 and at least 1, 2, 3, 4 or 5influenza subtypes of Group 2; (d) it treats or prevents infection by atleast 1, 2, 3, 4 or 5 influenza subtypes of Group 1, and by at least 1,2, 3, 4 or 5 influenza subtypes of Group 2; (e) it inhibits fusogenicactivity of the targeted HA; (f) it treats or prevents infection by aGroup 1 virus, wherein the virus is an H1, H5, or H9 virus; and treatsor prevents infection by a Group 2 virus, wherein the virus is an H3 orH7 virus; (g) it treats or prevents infection by influenza A strainsH1N1 and H3N2; (h) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H1N1 and H3N2 when administeredat 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2mg/kg or 1 mg/kg; (i) it treats or prevents infection by influenza Astrains H5N1; (j) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H5N1 when administered at 50mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kgor 1 mg/kg; (k) it binds with high affinity to a hemagglutinin (HA) ofan influenza B virus, e.g., B/Wisconsin/1/2010; (1) it treats orprevents infection by an influenza B virus, e.g., B/Wisconsin/1/2010;(m) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with an influenza B virus, e.g., B/Wisconsin/1/2010 whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (n) the concentration of antibody moleculerequired for 50% neutralization of influenza A virus is less than 10μg/mL; (o) the concentration of antibody molecule required for 50%neutralization of influenza B virus, e.g., B/Wisconsin/1/2010, is lessthan 10 μg/mL; (p) it prevents or minimizes secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject; (q) itis effective for preventing or minimizing secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (r) it binds an epitope which comprises orconsists of the hemagglutinin trimer interface; and (s) it binds anepitope other than that bound by a reference anti-HA antibody molecule,e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, e.g., whentested by a method disclosed herein, e.g., by competition in an ELISAassay.

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and S292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and 5329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

-   -   a and aa;    -   b and bb;    -   c and cc;    -   d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In another aspect, the disclosure features, a binding agent, e.g., anantibody molecule, or preparation, or isolated preparation thereof,comprising a structural or functional property of Ab 032.

In an embodiment, the antibody molecule competes with a referenceantibody molecule, e.g., an antibody molecule described herein, forbinding to a substrate, e.g., an HA. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A                (SEQ ID NO: 71);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:45; or    -   c) Ab 032.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004.Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g. from anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, which the reference antibody molecule binds. In anembodiment the antibody molecule does not bind to the same epitope, or aportion thereof, which the reference antibody molecule binds.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, on HA, as does a reference antibody molecule, e.g. anantibody molecule disclosed herein. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A                (SEQ ID NO: 71);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 25; and (ii) a light chain variable region segment        comprising SEQ ID NO:45; or    -   c) Ab 32.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004. Binding tothe same epitope, or a portion thereof, can be shown by one or more of:

-   -   a) mutational analysis, e.g., binding to HA, or binding affinity        for HA, is decreased or abolished if a residue is mutated;    -   b) analysis, e.g., comparison, of the crystal structure of the        antibody molecule and HA and the crystal structure of a        reference antibody and HA, e.g., to determine the touch points        of each;    -   c) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, from, e.g., an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004; and    -   d) (c) and one or both of (a) and (b);

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, from, e.g., anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45,

wherein each HC CDR differs by no more than 1, 2, 3, 4 or 5 amino acids,e.g., 1 or 2, e.g., conservative amino acids, from the corresponding CDRof SEQ ID NO: 25 and each LC CDR differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., 1 or 2, e.g., conservative amino acids, from thecorresponding CDR of SEQ ID NO: 45.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45,

wherein the antibody molecule comprises 1, 2, 3, 4, 5, or all of:

-   -   (i) a HC CDR1 comprising: S at the 1st position and A at the 3rd        position in HC CDR1;    -   (ii) a HC CDR2 comprising one or both, e.g., one of: V at the        2^(nd) position; or N at the 7^(th) position and Q at the 16th        position in HC CDR2;    -   (iii) a HC CDR3 comprising: R at the 3rd position (and        optionally, L at the 3^(rd) position);    -   (iv) a LC CDR1 comprising: I at the 3rd position;    -   (v) a LC CDR2 comprising one, two, or three of, e.g., one of: G        at the 2^(nd) position; Y at the 4th position; or L at the        5^(th) position in LC CDR2;    -   (vi) a LC CDR3 comprising: S at the 9^(th) position in LC CDR3;

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO:25 (or a sequence that differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., conservative amino acids, therefrom); and

(b) a light chain variable region segment comprising SEQ ID NO:155 (or asequence that differs by no more than 1, 2, 3, 4 or 5 amino acids, e.g.,conservative amino acids, therefrom).

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO: 25; and

(b) a light chain variable region segment comprising SEQ ID NO:155.

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);        and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence:        -   Q-S-I-T-F N-Y-K-N-Y-L-A (SEQ ID NO:71) (or a sequence that            differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2            amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom).

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a) LC CDR1-3, that collectively, differ from the AB 032 LC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids; and

b) HC CDR1-3, that collectively, differ from the AB 032 HC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids.

In an embodiment, the binding agent is an antibody molecule comprisingone or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, or 3, e.g., 1 or 2        amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1 or 2 of the highlighted        residues are not changed, e.g., both S and A are not changed);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom, provided that, e.g., at least 1, 2, or 3        of the highlighted residues are not changed, e.g., V or both N        and Q or all three of V, N, and Q are not changed);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom,        optionally provided that R is not changed); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence: Q-S-I-T-F-N-Y-K-N-Y-L-A (SEQ ID        NO: 71) or a sequence that differs by no more than, 1, 2, 3, 4,        or 5, e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom, optionally provided that at least 1 or 2 of the        highlighted residues are not changed, e.g., I is not changed);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1, 2, or 3 of the highlighted        residues are not changed, e.g., 1, 2 or all of G, Y, and L are        not changed);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom, optionally provided that at least one or both of the        highlighted residues are not changed, e.g., S is not changed).

In an embodiment a CDR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that CDR, (i.e., while other residues in that CDR might bechanged, the highlighted residue or combination of residues, are notchanged).

In an embodiment a CDR of the light and a CDR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In an embodiment each of two CDRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of the three CDRs in the heavy chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the three CDRs in the light chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the six CDRs in the heavy and light chainincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In one embodiment, the binding agent is an antibody molecule thatcomprises one or more or all of the following properties:

(a) both S and A in HC CDR1 are unchanged.

(b) V or both N and Q or all three of V, N, and Q in HC CDR2 areunchanged.

(c) R in HC CDR3 is unchanged.

(d) I in LC CDR1 is unchanged.

(e) 1, 2 or 3 of G, Y, and L in LC CDR2 are unchanged;

(f) S in LC CDR3 is unchanged.

In an embodiment the antibody molecule comprises 1, 2, 3, 4, 5, or all 6properties selected from (a) to (f).

In an embodiment, the antibody molecule comprises a heavy chain having aone or more properties selected from (a), (b), and (c) and a light chainhaving one or more properties selected from (d), (e), and (f).

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising:

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A (SEQ ID        NO: 71);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72); and    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73).

In some embodiments, the antibody molecule comprises one or more or allof the following properties: (i) it fails to produce any escape mutantsas determined by the failure of a viral titer to recover following atleast 10, 9, 8, 7, 6, or 5 rounds of serial infections in cell culturewith a mixture of the antibody molecule and an influenza virus (e.g., aninfluenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g.,A/South Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,or an H5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, oran influenza B virus, e.g., B/Wisconsin/1/2010); and (ii) it producesfewer escape mutants than does a reference anti-HA antibody molecule,such as Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, such as whentested by the method described in (i).

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from SEQ ID NO: 25. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 25; and

b) one or more framework regions (FRs) from SEQ ID NO: 45. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 45.

In one embodiment, the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable region segment that furthercomprises one or more or all of:

an FR1 comprising the sequenceQ-V-Q-L-L-E-T-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ IDNO:74) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that T is not changed);

an FR2 comprising the sequence W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ IDNO:75) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that W is not changed, or that if changed, is otherthan R);

an FR3 comprising the sequenceR-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K (SEQ IDNO:76) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2, amino acids, e.g., conservative amino acids, therefrom,optionally provided that one, two or three of I, R, or L is not changed,or that if I is changed it is other than G, if R is changed it is otherthan P. or if L is changed it is other than A); and

an FR4 comprising the sequence W-G-Q-G-T-T-L-T-V-S-S(SEQ ID NO:77) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom) orW-G-Q-G-T-T-V-T-V-S-S(SEQ ID NO:171) (or a sequence that differs by nomore than, 1, 2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g.,conservative amino acids, therefrom); and

(b) the light chain immunoglobulin variable region segment comprises oneor more or all of

an FR1 comprising the sequenceD-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C-R-S-S(SEQ ID NO:78) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom, optionallyprovided that R is not changed);

an FR2 comprising the sequence W-Y-Q-Q-K-P-G-K-A-P-K-L-L-I-Y (SEQ IDNO:79) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom);

an FR3 comprising the sequenceG-V-P-S-R-F-S-G-S-G-S-G-T-D-F-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C (SEQ IDNO:80) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that C is not changed, or if changed, is other thanP); and

an FR4 comprising the sequence F-G-Q-G-T-K-V-E-I-K (SEQ ID NO:81) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom).

In an embodiment a FR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that FR, (i.e., while other residues in that FR might bechanged, the highlighted residue or combination of residues, are notchanged). E.g., in an embodiment, one, two or three of I, R, or L forheavy chain FR3 is not changed.

In an embodiment a FR of the light and a FR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that FR.

In an embodiment each of two FRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that FR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of FR2 and FR3 in the heavy chain includes one ofthe highlighted residues, or one of the highlighted combinations ofresidues, for that FR.

In an embodiment each of FR1 and FR2 in the heavy and light chainincludes one of the highlighted residues for that FR.

In an embodiment all of the highlighted residues in heavy chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in light chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in both heavy and lightchain FR1-4 are unchanged.

In another embodiment, the binding agent, e.g., an antibody molecule,comprises one or more or all of the following properties: (a) it failsto produce any escape mutants as determined by the failure of a viraltiter to recover following at least 10, 9, 8, 7, 6, or 5 rounds ofserial infections in cell culture with a mixture of the antibodymolecule and an influenza virus (e.g., an influenza A virus, e.g., aGroup 1 strain, e.g., an H1N1 strain, e.g., A/South Carolina/1/1918,A/Puerto Rico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., B/Wisconsin/1/2010); (b) it produces fewer escape mutants thandoes a reference anti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28,C179, or CR6261, e.g., when tested by the method described in (a); (c)it binds with high affinity to a hemagglutinin (HA) of at least 1, 2, 3,4 or 5 influenza subtypes of Group 1 and at least 1, 2, 3, 4 or 5influenza subtypes of Group 2; (d) it treats or prevents infection by atleast 1, 2, 3, 4 or 5 influenza subtypes of Group 1, and by at least 1,2, 3, 4 or 5 influenza subtypes of Group 2; (e) it inhibits fusogenicactivity of the targeted HA; (f) it treats or prevents infection by aGroup 1 virus, wherein the virus is an H1, H5, or H9 virus; and treatsor prevents infection by a Group 2 virus, wherein the virus is an H3 orH7 virus; (g) it treats or prevents infection by influenza A strainsH1N1 and H3N2; (h) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H1N1 and H3N2 when administeredat 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2mg/kg or 1 mg/kg; (i) it treats or prevents infection by influenza Astrains H5N1; (j) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with H5N1 when administered at 50mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kgor 1 mg/kg; (k) it binds with high affinity to a hemagglutinin (HA) ofan influenza B virus, e.g., B/Wisconsin/1/2010; (1) it treats orprevents infection by an influenza B virus, e.g., B/Wisconsin/1/2010;(m) it is effective for prevention or treatment of infection, e.g., inhumans or mice, with an influenza B virus, e.g., B/Wisconsin/1/2010 whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (n) the concentration of antibody moleculerequired for 50% neutralization of influenza A virus is less than 10μg/mL; (o) the concentration of antibody molecule required for 50%neutralization of influenza B virus, e.g., B/Wisconsin/1/2010, is lessthan 10 μg/mL; (p) it prevents or minimizes secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject; (q) itis effective for preventing or minimizing secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg or 1 mg/kg; (r) it binds an epitope which comprises orconsists of the hemagglutinin trimer interface; and (s) it binds anepitope other than that bound by a reference anti-HA antibody molecule,e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, e.g., whentested by a method disclosed herein, e.g., by competition in an ELISAassay.

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and S292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and S329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

-   -   a and aa;    -   b and bb;    -   c and cc;    -   d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In another aspect, the disclosure features, a binding agent, e.g., anantibody molecule, or preparation, or isolated preparation thereof,comprising a structural or functional property of Ab 031.

In an embodiment, the antibody molecule competes with a referenceantibody molecule, e.g., an antibody molecule described herein, forbinding to a substrate, e.g., an HA. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A                (SEQ ID NO:71);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 24; and (ii) a light chain variable region segment        comprising SEQ ID NO:45; or    -   c) Ab 031.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004.Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g. from anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, which the reference antibody molecule binds.

In an embodiment the antibody molecule does not bind to the sameepitope, or a portion thereof, which the reference antibody moleculebinds.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, on HA, as does a reference antibody molecule, e.g. anantibody molecule disclosed herein. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising:        -   i) a heavy chain immunoglobulin variable region segment            comprising            -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);            -   a CDR2 comprising the sequence                V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G            -   (SEQ ID NO:69); and            -   a CDR3 comprising the sequence                D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70);                and        -   ii) a light chain variable region segment comprising:            -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A                (SEQ ID NO:71);            -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID                NO:72); and            -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID                NO:73).    -   b) an antibody molecule comprises one or both of: (i) a heavy        chain immunoglobulin variable region segment comprising SEQ ID        NO: 24; and (ii) a light chain variable region segment        comprising SEQ ID NO:45; or    -   c) Ab 031.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004. Binding tothe same epitope, or a portion thereof, can be shown by one or more of:

-   -   a) mutational analysis, e.g., binding to HA, or binding affinity        for HA, is decreased or abolished if a residue is mutated;    -   b) analysis, e.g., comparison, of the crystal structure of the        antibody molecule and HA and the crystal structure of a        reference antibody and HA, e.g., to determine the touch points        of each;    -   c) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, from, e.g., an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004;    -   d) (c) and one or both of (a) and (b).

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, from, e.g., anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry.

The antibody molecule can compete with the reference antibody such thatbinding of the reference antibody is decreased by 50% or more.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 24;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 24;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45,

wherein, optionally, each HC CDR differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., 1 or 2, e.g., conservative amino acids, from thecorresponding CDR of SEQ ID NO: 24 and each LC CDR differs by no morethan 1, 2, 3, 4 or 5 amino acids, e.g., 1 or 2, e.g., conservative aminoacids, from the corresponding CDR of SEQ ID NO: 45.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with SEQ ID NO: 25;

and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with SEQ ID NO: 45,

wherein the antibody molecule comprises 1, 2, 3, 4, 5, or all of:

-   -   (i) a HC CDR1 comprising: S at the 1st position and A at the 3rd        position in HC CDR1;    -   (ii) a HC CDR2 comprising one or both, e.g., one of: V at the        2nd position; or N at the 7^(th) position and Q at the 16^(th)        position in HC CDR2;    -   (iii) a HC CDR3 comprising: R at the 3rd position (and        optionally, L at the 3^(rd) position);    -   (iv) a LC CDR1 comprising: I at the 3rd position;    -   (v) a LC CDR2 comprising one, two, or three of, e.g., one of: G        at the 2nd position; Y at the 4^(th) position; or L at the        5^(th) position in LC CDR2;    -   (vi) a LC CDR3 comprising: S at the 9^(th) position in LC CDR3;

In an embodiment, the binding agent comprises an antibody moleculecomprising:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO:24(or a sequence that differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., conservative amino acids, therefrom); and

(b) a light chain variable region segment comprising SEQ ID NO:45 (or asequence that differs by no more than 1, 2, 3, 4 or 5 amino acids, e.g.,conservative amino acids, therefrom).

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising SEQID NO: 24; and

(b) a light chain variable region segment comprising SEQ ID NO:45.

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom); and    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom);        and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A (SEQ ID        NO:71) (or a sequence that differs by no more than, 1, 2, 3, 4,        or 5, e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO: 72) (or        a sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g.,        1 or 2 amino acids, e.g., conservative amino acids, therefrom);        and    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom).

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a) LC CDR1-3, that collectively, differ from the AB 031 LC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids; and

b) HC CDR1-3, that collectively, differ from the AB 031 HC CDR1-3 by nomore than, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, 3, or 4, aminoacids, e.g., conservative amino acids.

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

-   -   (a) a heavy chain immunoglobulin variable region segment        comprising    -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68) (or a        sequence that differs by no more than, 1, 2, or 3, e.g., 1 or 2        amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1 or 2 of the highlighted        residues are not changed, e.g., both S and A are not changed);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69) (or a sequence that differs by no more than, 1,        2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g., conservative        amino acids, therefrom, provided that, e.g., at least 1, 2, or 3        of the highlighted residues are not changed, e.g., V or both N        and Q or all three of V, N, and Q are not changed);    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom        optionally provided that, e.g., R is not changed); and    -   (b) a light chain variable region segment comprising    -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A (SEQ ID        NO: 71) or a sequence that differs by no more than, 1, 2, 3, 4,        or 5, e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom, optionally provided that at least 1 or 2 of the        highlighted residues are not changed, e.g., I is not changed);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72) (or a        sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1        or 2 amino acids, e.g., conservative amino acids, therefrom,        optionally provided that at least 1, 2, or 3 of the highlighted        residues are not changed, e.g., 1, 2 or all of G, Y, and L are        not changed);    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S (SEQ ID NO:73)        (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,        e.g., 1 or 2 amino acids, e.g., conservative amino acids,        therefrom, optionally provided that at least one or both of the        highlighted residues are not changed, e.g., S is not changed).

In an embodiment a CDR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that CDR, (i.e., while other residues in that CDR might bechanged, the highlighted residue or combination of residues, are notchanged).

In an embodiment a CDR of the light and a CDR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In an embodiment each of two CDRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of the three CDRs in the heavy chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the three CDRs in the light chain includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that CDR.

In an embodiment each of the six CDRs in the heavy and light chainincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that CDR.

In one embodiment, the binding agent is an antibody molecule thatcomprises one or more or all of the following properties:

(a) both S and A in HC CDR1 are unchanged.

(b) V or both N and Q or all three of V, N, and Q in HC CDR2 areunchanged.

(c) R in HC CDR3 is unchanged.

(d) I in LC CDR1 is unchanged.

(e) 1, 2 or 3 of G, Y, and L in LC CDR2 are unchanged;

(f) S in LC CDR3 is unchanged.

In an embodiment the antibody molecule comprises 1, 2, 3, 4, 5, or all 6properties selected from (a) to (f).

In an embodiment, the antibody molecule comprises a heavy chain having aone or more properties selected from (a), (b), and (c) and a light chainhaving one or more properties selected from (d), (e), and (f).

In the embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising

-   -   a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68);    -   a CDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G        (SEQ ID NO:69); and    -   a CDR3 comprising the sequence        D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70); and

(b) a light chain variable region segment comprising

-   -   a CDR1 comprising the sequence Q-S-I-T-F-N-Y-K-N-Y-L-A (SEQ ID        NO:71);    -   a CDR2 comprising the sequence W-G-S-Y-L-E-S(SEQ ID NO:72); and    -   a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-S(SEQ ID NO:73).

In some embodiments, the antibody molecule comprises one or more or allof the following properties: (i) it fails to produce any escape mutantsas determined by the failure of a viral titer to recover following atleast 10, 9, 8, 7, 6, or 5 rounds of serial infections in cell culturewith a mixture of the antibody molecule and an influenza virus (e.g., aninfluenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g.,A/South Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,or an H5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, oran influenza B virus, e.g., B/Wisconsin/1/2010); and (ii) it producesfewer escape mutants than does a reference anti-HA antibody molecule,e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, e.g., whentested by the method described in (i).

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from SEQ ID NO: 24. E.g., theantibody molecule comprises one or more or all of FR1, FR2, 1-R3, orFR4, or sequences that differ individually, or collectively, by no morethan 1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues,from SEQ ID NO: 24; and

b) one or more framework regions (FRs) from SEQ ID NO: 45. E.g., theantibody molecule comprises one or more or all of FR1, FR2, FR3, or FR4,or sequences that differ individually, or collectively, by no more than1, 2, 3, 4, of 5 amino acid residues, e.g., conservative residues, fromSEQ ID NO: 45.

In one embodiment, the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable region segment that furthercomprises one or more or all of:

an FR1 comprising the sequenceE-V-Q-L-L-E-S-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ IDNO:82) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that T is not changed);

an FR2 comprising the sequence W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ IDNO:75) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that W is not changed, or that if changed, is otherthan R);

an FR3 comprising the sequenceR-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K (SEQ IDNO:76) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that one, two or three of I, R, or L is not changed,or that if I is changed it is other than G, if R is changed it is otherthan P. or if L is changed it is other than A); and

an FR4 comprising the sequence W-G-Q-G-T-T-L-T-V-S-S (SEQ ID NO:77) (ora sequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom) orW-G-Q-G-T-T-V-T-V-S-S (SEQ ID NO:171) (or a sequence that differs by nomore than, 1, 2, 3, 4, or 5, e.g., 1 or 2 amino acids, e.g.,conservative amino acids, therefrom); and

(a) a light chain immunoglobulin variable region segment furthercomprises one or more or all of:

an FR1 comprising the sequenceD-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C-R-S-S(SEQ ID NO:78) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom, optionallyprovided that R is not changed);

an FR2 comprising the sequence W-Y-Q-Q-K-P-G-K-A-P-K-L-L-I-Y (SEQ IDNO:79) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom);

an FR3 comprising the sequenceG-V-P-S-R-F-S-G-S-G-S-G-T-D-F-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C (SEQ IDNO:80) (or a sequence that differs by no more than, 1, 2, 3, 4, or 5,e.g., 1 or 2 amino acids, e.g., conservative amino acids, therefrom,optionally provided that C is not changed, or if changed, is other thanP); and

an FR4 comprising the sequence F-G-Q-G-T-K-V-E-I-K (SEQ ID NO:81) (or asequence that differs by no more than, 1, 2, 3, 4, or 5, e.g., 1 or 2amino acids, e.g., conservative amino acids, therefrom).

In an embodiment a FR of the light or heavy chain includes one of thehighlighted residues, or one of the highlighted combinations ofresidues, for that FR, (i.e., while other residues in that FR might bechanged, the highlighted residue or combination of residues, are notchanged). E.g., in an embodiment, one, two or three of I, R, or L forheavy chain FR3 is not changed.

In an embodiment a FR of the light and a FR of the heavy chain eachincludes one of the highlighted residues, or one of the highlightedcombinations of residues, for that FR.

In an embodiment each of two FRs in the antibody molecule includes oneof the highlighted residues, or one of the highlighted combinations ofresidues, for that FR. In embodiments both are in the light chain. Inembodiments both are in the heavy chain.

In an embodiment each of FR2 and FR3 in the heavy chain includes one ofthe highlighted residues, or one of the highlighted combinations ofresidues, for that FR.

In an embodiment each of FR1 and FR2 in the heavy and light chainincludes one of the highlighted residues for that FR.

In an embodiment all of the highlighted residues in heavy chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in light chain FR1-4are unchanged.

In an embodiment all of the highlighted residues in both heavy and lightchain FR1-4 are unchanged.

In one embodiment, the antibody molecule comprises:

(a) the heavy chain immunoglobulin variable region segment comprises oneor more or all of

-   -   an FR1 comprising the sequence        E-V-Q-L-L-E-S-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ        ID NO:82);    -   an FR2 comprising the sequence W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ        ID NO:75);    -   an FR3 comprising the sequence        R-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K        (SEQ ID NO:76); and    -   an FR4 comprising the sequence W-G-Q-G-T-T-L-T-V-S-S (SEQ ID        NO:77) or W-G-Q-G-T-T-V-T-V-S-S (SEQ ID NO:171); and

(b) the light chain immunoglobulin variable region segment comprisingone or more or all of

-   -   an FR1 comprising the sequence        D-I-Q-M-T-Q-S-P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C-R-S-S (SEQ ID        NO:78);    -   an FR2 comprising the sequence W-Y-Q-Q-K-P-G-K-A-P-K-L-L-I-Y        (SEQ ID NO:79);    -   an FR3 comprising the sequence        G-V-P-S-R-F-S-G-S-G-S-G-T-D-F-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C        (SEQ ID NO:80); and    -   an FR4 comprising the sequence F-G-Q-G-T-K-V-E-I-K (SEQ ID        NO:81).

In another embodiment, the antibody molecule comprises one or more orall of the following properties: (a) it fails to produce any escapemutants as determined by the failure of a viral titer to recoverfollowing at least 10, 9, 8, 7, 6, or 5 rounds of serial infections incell culture with a mixture of the antibody molecule and an influenzavirus (e.g., an influenza A virus, e.g., a Group 1 strain, e.g., an H1N1strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004, or an influenza B virus, e.g., B/Wisconsin/1/2010);(b) it produces fewer escape mutants than does a reference anti-HAantibody molecule, e.g., Ab 67-11, F16, F128, C179, F10, CR9114, orCR6261, e.g., when tested by the method described in (a); (c) it bindswith high affinity to a hemagglutinin (HA) of at least 1, 2, 3, 4 or 5influenza subtypes of Group 1 and at least 1, 2, 3, 4 or 5 influenzasubtypes of Group 2; (d) it treats or prevents infection by at least 1,2, 3, 4 or 5 influenza subtypes of Group 1, and by at least 1, 2, 3, 4or 5 influenza subtypes of Group 2; (e) it inhibits fusogenic activityof the targeted HA; (f) it treats or prevents infection by a Group 1virus, wherein the virus is an H1, H5, or H9 virus; and treats orprevents infection by a Group 2 virus, wherein the virus is an H3 or H7virus; (g) it treats or prevents infection by influenza A strains H1N1and H3N2; (h) it is effective for prevention or treatment of infection,e.g., in humans or mice, with H1N1 and H3N2 when administered at 50mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg,or 1 mg/kg; (i) it treats or prevents infection by influenza A strainsH5N1; (j) it is effective for prevention or treatment of infection,e.g., in humans or mice, with H5N1 when administered at 50 mg/kg, 25mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1mg/kg; (k) it binds with high affinity to a hemagglutinin (HA) of aninfluenza B virus, e.g., B/Wisconsin/1/2010; (1) it treats or preventsinfection by an influenza B virus, e.g., B/Wisconsin/1/2010; (m) it iseffective for prevention or treatment of infection, e.g., in humans ormice, with an influenza B virus, e.g., B/Wisconsin/1/2010 whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg, or 1 mg/kg; (n) the concentration of antibody moleculerequired for 50% neutralization of influenza A virus is less than 10μg/mL; (o) the concentration of antibody molecule required for 50%neutralization of influenza B virus, e.g., B/Wisconsin/1/2010, is lessthan 10 μg/mL; (p) it prevents or minimizes secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject; (q) itis effective for preventing or minimizing secondary infection (e.g.,secondary bacterial infection) or effects thereof on a subject whenadministered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg,3 mg/kg, 2 mg/kg, or 1 mg/kg; (r) it binds an epitope which comprises orconsists of the hemagglutinin trimer interface; and (s) it binds anepitope other than that bound by a reference anti-HA antibody molecule,e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, e.g., whentested by a method disclosed herein, e.g., by competition in an ELISAassay.

In another aspect, the disclosure features an antibody moleculecomprising: (a) a heavy chain immunoglobulin variable region segmentcomprising SEQ ID NO:24 (or a sequence that differs by no more than 1,2, 3, 4 or 5 amino acids, e.g., conservative amino acids, therefrom);and (b) a light chain variable region segment comprising SEQ ID NO:45(or a sequence that differs by no more than 1, 2, 3, 4 or 5 amino acids,e.g., conservative amino acids, therefrom). In some embodiments, theantibody molecule comprises one or more or all of the followingproperties: (i) it fails to produce any escape mutants as determined bythe failure of a viral titer to recover following at least 10, 9, 8, 7,6, or 5 rounds of serial infections in cell culture with a mixture ofthe antibody molecule and an influenza a virus, e.g., a Group 1 strain,e.g., an H1N1 strain, e.g., A/South Carolina/1/1918, A/PuertoRico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004; and (ii) it produces fewerescape mutants than does a reference anti-HA antibody molecule, such asAb 67-11, FI6, FI28, C179, F10, CR9114, or CR6261, such as when testedby the method described in (i).

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and S292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and S329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

-   -   a and aa;    -   b and bb;    -   c and cc;    -   d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In another aspect, the disclosure features, a binding agent, e.g., anantibody molecule, or preparation, or isolated preparation thereof,comprising a structural or functional property of one or both a heavychain variable region and a light chain variable region disclosedherein.

In an embodiment, the antibody molecule competes with a referenceantibody molecule, e.g., an antibody molecule described herein, forbinding to a substrate, e.g., an HA. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising the heavy and light CDRs        from:

a heavy chain variable region from Table 3, Table 4A, Table 4B, FIG. 2,FIG. 13, or FIG. 17; and

a light chain variable region from Table 3, Table 4A, Table 4B, FIG. 3,FIG. 14, or FIG. 17.

-   -   b) an antibody molecule that comprises: (i) a heavy chain        immunoglobulin variable region segment from Table 3, Table 4A,        Table 4B, FIG. 2, FIG. 13, or FIG. 17; and (ii) a light chain        variable region segment from Table 3, Table 4A, Table 4B, FIG.        3, FIG. 14, or FIG. 17; or    -   c) an antibody disclosed herein.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004.Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, e.g. from anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry. The antibody molecule can compete with the        reference antibody such that binding of the reference antibody        is decreased by 50% or more. In an embodiment the antibody        molecule binds to the same epitope, or a portion thereof, which        the reference antibody molecule binds. In an embodiment the        antibody molecule does not bind to the same epitope, or a        portion thereof, which the reference antibody molecule binds.

In an embodiment the antibody molecule binds to the same epitope, or aportion thereof, on HA, as does a reference antibody molecule, e.g. anantibody molecule disclosed herein. The reference antibody molecule canbe:

-   -   a) an antibody molecule comprising the heavy and light CDRs        from:

a heavy chain variable region from Table 3, Table 4A, Table 4B, FIG. 2,FIG. 13, or FIG. 17; and

a light chain variable region from Table 3, Table 4A, Table 4B, FIG. 3,FIG. 14, or FIG. 17.

-   -   b) an antibody molecule that comprises: (i) a heavy chain        immunoglobulin variable region segment from Table 3, Table 4A,        Table 4B, FIG. 2, FIG. 13, or FIG. 17; and (ii) a light chain        variable region segment from Table 3, Table 4A, Table 4B, FIG.        3, FIG. 14, or FIG. 17; or    -   c) an antibody disclosed herein.

The HA can be HA1 or HA5, e.g. from an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004. Binding tothe same epitope, or a portion thereof, can be shown by one or more of:

-   -   a) mutational analysis, e.g., binding to HA, or binding affinity        for HA, is decreased or abolished if a residue is mutated;    -   b) analysis, e.g., comparison, of the crystal structure of the        antibody molecule and HA and the crystal structure of a        reference antibody and HA, e.g., to determine the touch points        of each;    -   c) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, e.g. from an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004; and    -   d) (c) and one or both of (a) and (b);

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, from, e.g., anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art. Reduction of the ability to bind can beevaluated, e.g., by one or more of:

-   -   a) BIAcore analysis;    -   b) ELISA assay; and    -   c) flow cytometry. The antibody molecule can compete with the        reference antibody such that binding of the reference antibody        is decreased by 50% or more.    -   d) competition of the two antibodies for binding to HA, e.g.,        HA1 or HA5, from, e.g., an H1N1 strain, e.g., A/South        Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,        or an H5N1 strain, e.g., A/Indonesia/5/2005 or        A/Vietnam/1203/2004; and    -   e) (c) and one or both of (a) and (b).

Competition between the antibody molecule and a reference antibodymolecule can be determined by evaluating the ability of one of theantibody molecule or the reference antibody molecule to decrease bindingof the other to a substrate, e.g., HA, e.g., HA1 or HA5, from, e.g., anH1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. Reduction of the ability to bind can be evaluatedby methods in the art.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with a reference heavy chain from Table 3,Table 4A, Table 4B, FIG. 2, FIG. 13 or FIG. 17; and

a light chain variable region comprising least 60, 70, 80, 85, 90, 95,98 or 99 percent homology with reference light chain from Table 3, Table4A, Table 4B, FIG. 3, FIG. 14 or FIG. 17,

wherein, optionally, each HC CDR differs by no more than 1, 2, 3, 4 or 5amino acids, e.g., 1 or 2, e.g., conservative amino acids, from thecorresponding HC CDR from its reference heavy chain and each LC CDRdiffers by no more than 1, 2, 3, 4 or 5 amino acids, e.g., 1 or 2, e.g.,conservative amino acids, from the corresponding CDR in its referencelight chain.

In an embodiment the binding agent, e.g., an antibody molecule,comprises: a heavy chain variable region comprising least 60, 70, 80,85, 90, 95, 98 or 99 percent homology with a heavy chain from Table 3and a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with the corresponding light chain fromTable 3.

In an embodiment the binding agent, e.g., an antibody molecule,comprises: a heavy chain variable region comprising least 60, 70, 80,85, 90, 95, 98 or 99 percent homology with a heavy chain from Table 4Aand a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with the corresponding light chain fromTable 4A.

In an embodiment the binding agent, e.g., an antibody molecule,comprises: a heavy chain variable region comprising least 60, 70, 80,85, 90, 95, 98 or 99 percent homology with a heavy chain from Table 4Band a light chain variable region comprising least 60, 70, 80, 85, 90,95, 98 or 99 percent homology with the corresponding light chain fromTable 4B.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

a heavy chain variable region from Table 3, Table 4A, Table 4B, FIG. 2,FIG. 13, or FIG. 17; and

a light chain variable region from Table 3, Table 4A, Table 4B, FIG. 3,FIG. 14, or FIG. 17.

In an embodiment the binding agent, e.g., an antibody molecule,comprises:

a heavy chain variable region from Table 3 and the corresponding lightchain from Table 3;

a heavy chain from Table 4A and the corresponding light chain from Table4A; or

a heavy chain from Table 4B and the corresponding light chain from Table4B.

In an embodiment, the binding agent, e.g., an antibody molecule,comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising aCDR1, a CDR2 and a CDR3 from a heavy chain sequence of Table 3, Table4A, Table 4B, FIG. 2, FIG. 13, or FIG. 17 (or CDRs that, individually orcollectively, differ therefrom by no more than, 1, 2, 3, 4, or 5, e.g.,1 or 2 amino acids, e.g., conservative amino acids)); and

(b) a light chain immunoglobulin variable region segment comprising aCDR1, a CDR2 and a CDR3 from a light chain sequence of Table 3, Table4A, Table 4B, FIG. 3, FIG. 14, or FIG. 17 (or CDRs that, individually orcollectively, differ therefrom by no more than, 1, 2, 3, 4, or 5, e.g.,1 or 2 amino acids, e.g., conservative amino acids).

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

CDRs from a heavy chain of Table 3 and the light chain CDRs from thecorresponding light chain from Table 3.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

CDRs from a heavy chain of Table 4A and the light chain CDRs from thecorresponding light chain from Table 4A.

In an embodiment the binding agent, e.g., an antibody molecule,comprises one or both of:

CDRs from a heavy chain of Table 4B and the light chain CDRs from thecorresponding light chain from Table 4B.

In some embodiments, the binding agent, e.g., an antibody molecule,comprises one or more or all of the following properties: (i) it failsto produce any escape mutants as determined by the failure of a viraltiter to recover following at least 10, 9, 8, 7, 6, or 5 rounds ofserial infections in cell culture with a mixture of the antibodymolecule and an influenza virus (e.g., an influenza A virus, e.g., aGroup 1 strain, e.g., an H1N1 strain, e.g., A/South Carolina/1/1918,A/Puerto Rico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., B/Wisconsin/1/2010); (ii) it produces fewer escape mutants thandoes a reference anti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28,C179, F10, CR9114, or CR6261, e.g., when tested by the method describedin (i); and (iii) it is other than Ab 67-11 and FI6.

In one embodiment, the antibody molecule comprises one or both of:

(a) a heavy chain immunoglobulin variable region segment comprising aCDR1, a CDR2; and a CDR3 from a heavy chain sequence of FIG. 2, FIG. 13,or FIG. 17; and

(b) a light chain immunoglobulin variable region segment comprising aCDR1, a CDR2 and a CDR3 from a light chain sequence of FIG. 3, FIG. 14,or FIG. 17.

In one embodiment, the antibody molecule comprises:

(a) a heavy chain immunoglobulin variable region segment from FIG. 2 orFIG. 17; and

(b) a light chain immunoglobulin variable region segment from FIG. 3 orFIG. 17.

In one embodiment, the heavy chain immunoglobulin variable regionfurther comprises an Isoleucine-Aspartate (Ile-Asp) dipeptide at theN-terminus. In another embodiment, the light chain immunoglobulinvariable region further comprises an Ile-Asp dipeptide at theN-terminus. In yet another embodiment, both the heavy chainimmunoglobulin variable region and the light chain immunoglobulinvariable region or an antibody featured in the disclosure furthercomprises an Ile-Asp dipeptide at the N-terminus. In other embodimentthe Ile-Asp dipeptide is absent from one or both the heavy and lightchain.

In one embodiment, the binding agent, e.g., an antibody molecule,further comprises one or more or all of the following: (a) it treats orprevents infection by at least 1, 2, 3, 4 or 5 influenza subtypes ofGroup 1, and by at least 1, 2, 3, 4 or 5 influenza subtypes of Group 2;(b) it inhibits fusogenic activity of the targeted HA; (c) it treats orprevents infection by a Group 1 virus, wherein the virus is an H1, H5,or H9 virus; and treats or prevents infection by a Group 2 virus,wherein the virus is an H3 or H7 virus; (d) it treats or preventsinfection by influenza A strains H1N1 and H3N2; (e) it is effective forprevention or treatment of infection, e.g., in humans or mice, with H1N1and H3N2 when administered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg; (f) it treats or preventsinfection by influenza A strains H5N1; (g) it is effective forprevention or treatment of infection, e.g., in humans or mice, with H5N1when administered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6 mg/kg, 5 mg/kg, 4mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg; (h) it binds with high affinity toa hemagglutinin (HA) of an influenza B virus, e.g., B/Wisconsin/1/2010;(i) it treats or prevents infection by an influenza B virus, e.g.,B/Wisconsin/1/2010; (j) it is effective for prevention or treatment ofinfection, e.g., in humans or mice, with an influenza B virus, e.g.,B/Wisconsin/1/2010 when administered at 50 mg/kg, 25 mg/kg, 10 mg/kg, 6mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg; (k) theconcentration of antibody molecule required for 50% neutralization ofinfluenza A virus is less than 10 μg/mL; (1) the concentration ofantibody molecule required for 50% neutralization of influenza B virus,e.g., B/Wisconsin/1/2010, is less than 10 μg/mL; (m) it prevents orminimizes secondary infection (e.g., secondary bacterial infection) oreffects thereof on a subject; (n) it is effective for preventing orminimizing secondary infection (e.g., secondary bacterial infection) oreffects thereof on a subject when administered at 50 mg/kg, 25 mg/kg, 10mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg; (o) itbinds an epitope which comprises or consists of the hemagglutinin trimerinterface; and (p) it binds an epitope other than that bound by areference anti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28, C179,F10, CR9114, or CR6261, e.g., when tested by a method disclosed herein,e.g., by competition in an ELISA assay.

In an embodiment the antibody molecule comprises one or both of:

a) one or more framework regions (FRs) from heavy chain disclosedherein. E.g., the antibody molecule comprises one or more or all of FR1,FR2, FR3, or FR4, or sequences that differ individually, orcollectively, by no more than 1, 2, 3, 4, of 5 amino acid residues,e.g., conservative residues, from heavy chain disclosed herein; and

b) one or more framework regions (FRs) from light chain disclosedherein. E.g., the antibody molecule comprises one or more or all of FR1,FR2, FR3, or FR4, or sequences that differ individually, orcollectively, by no more than 1, 2, 3, 4, of 5 amino acid residues,e.g., conservative residues, from light chain disclosed herein.

In an embodiment the binding agent, e.g., an antibody molecule,specifically binds the HA antigen.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   a) it includes one, two, or all of, H3 HA1 residues N38, I278,        and D291;    -   b) it includes H3 HA2 residue N12;    -   c) it does not include one, two or all of, H3 HA1 residues Q327,        T328, and R329;    -   d) it does not include one, two, three, four, or all of, H3 HA2        residues G1, L2, F3, G4, and D46;    -   e) it includes one, two, or all of, H3 HA1 residues T318, R321,        and V323; or    -   f) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H3 HA2 residues A7, E11, I18, D19, G20,        W21, L38, K39, T41, Q42, A43, I45, I48, N49, L52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: a; and b.

In an embodiment the antibody molecule has properties: c; and d.

In an embodiment the antibody molecule has properties: a; and c or d.

In an embodiment the antibody molecule has properties: b; and c or d.

In an embodiment the antibody molecule has properties: c; and a or b.

In an embodiment the antibody molecule has properties: d; and a or b.

In an embodiment the antibody molecule has properties: a, b, c and d.

In an embodiment the antibody molecule has properties: a, b, c, d, e,and f.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   a) H3 HA1 residues N38, I278, or D291;    -   b) H3 HA2 residue N12;    -   c) H3 HA1 residues T318, R321, or V323; or    -   d) H3 HA2 residues A7, E11, I18, D19, G20, W21, L38, K39, T41,        Q42, A43, I45, I48, N49, L52, N53, I56, or E57.

In an embodiment, the antibody molecule has a K_(D) for H3 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   c) H3 HA1 residues Q327, T328, or R329; or    -   d) H3 HA2 residues G1, L2, F3, G4, or D46.

In an embodiment, the antibody molecule binds an epitope that has one,two, three, four, five, or all of, the following properties a-f:

-   -   aa) it includes one, two, or all of, H1 HA1 residues H31, N279,        and S292;    -   bb) it includes H1 HA2 residue G12;    -   cc) it does not include one or both of H1 HA1 residues Q328 and        S329;    -   dd) it does not include one, two, three, four, or all of, H1 HA2        residues G1, L2, F3, G4, and D46;    -   ee) it includes one, two, or all of, H1 HA1 residues T319, R322,        and I324 are bound by both Ab 044 and FI6; or    -   ff) it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, or all of, H1 HA2 residues A7, E11, I18, D19, G20,        W21, Q38, K39, T41, Q42, N43, I45, I48, T49, V52, N53, I56, and        E57.

In an embodiment the antibody molecule has properties: aa; and bb.

In an embodiment the antibody molecule has properties: cc; and dd.

In an embodiment the antibody molecule has properties: aa; and cc or dd.

In an embodiment the antibody molecule has properties: bb; and cc or dd.

In an embodiment the antibody molecule has properties: cc; and aa or bb.

In an embodiment the antibody molecule has properties: dd; and aa or bb.

In an embodiment the antibody molecule has properties: aa, bb, cc anddd.

In an embodiment the antibody molecule has properties: aa, bb, cc, dd,ee, and ff.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by at least 2, 5, 10,or 100 fold, by a mutation or mutations in any of:

-   -   aa) H1 HA1 residues H31, N279, and 5292;    -   bb) H1 HA2 residue G12;    -   cc) H1 HA1 residues T319, R322, and I324; or    -   dd) H1 HA2 residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,        Q42, N43, I45, I48, T49, V52, N53, I56, and E57.

In an embodiment, the antibody molecule has a K_(D) for H1 of equal toor less than 10⁻⁶, wherein said K_(D) is increased by no more than 2, or5 fold, by a mutation or mutations in any of:

-   -   cc) H1 HA1 residues Q328 and 5329; or    -   dd) H1 HA2 residues G1, L2, F3, G4, and D46;

In an embodiment the antibody molecule has one, two, three or all of thefollowing properties:

-   -   a and aa;    -   b and bb;    -   c and cc;    -   d and dd.

In an embodiment the molecule has properties c, cc, d, and dd.

In one aspect, the disclosure features an anti-hemagglutinin (anti-HA)binding agent, e.g., antibody molecule, or preparation, or isolatedpreparation thereof, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising oneor more or all of a

CDR1 comprising the sequence G-F-T-F-[S/T]-[S/T]-Y-[A/G]-M-H (SEQ IDNO:184), or a sequence that differs from SEQ ID NO:184 by no more than 1or 2 residues;

a CDR2 comprising the sequenceV-[I/V/L]-S-[Y/F]-D-G-[S/N]-[Y/N]-[K/R]-Y-Y-A-D-S-V-Q-G (SEQ ID NO:2) ora sequence that differs from SEQ ID NO:2 by no more than 1 or 2residues; and

a CDR3 comprising the sequenceD-[S/T]-[R/K/Q]-L-R-[S/T]-L-L-Y-F-E-W-L-S-[Q/S]-G-[Y/L/V]-[F/L]-[N/D]-[P/Y](SEQ ID NO:3), or a sequence that differs from SEQ ID NO:3 by no morethan 1 or 2 residues; and

(b) a light chain variable region segment comprising one or more or allof a CDR1 comprising the sequence[K/R]-S-S-Q-[S/T]-[V/L/I]-[T/S]-[Y/F/W]-[N/S/D]-Y-K-N-Y-L-A (SEQ IDNO:185) or a sequence that differs from SEQ ID NO:185 by no more than 1or 2 residues, or comprising the sequence[K/R]-S-S-Q-[S/T]-[V/L/I]-[T/S]-[Y/F/W]-[N/S/D/Q/R/E]-Y-K-N-Y-L-A (SEQID NO:186) or a sequence that differs from SEQ ID NO:186 by no more than1 or 2 residues or[K/R]-S-S-Q-[S/T]-[V/L/I]-[T/S]-[Y/F/W]-[N/S/D/E]-Y-K-N-Y-L-A (SEQ IDNO: 185) or a sequence that differs from SEQ ID NO:185 by no more than 1or 2 residues;

a CDR2 comprising the sequence W-[A/G]-S-[T/A/Y/H/K/D]-[R/L]-E-[S/T](SEQ ID NO:5) or a sequence that differs from SEQ ID NO:5 by no morethan 1 or 2 residues;

a CDR3 comprising the sequence Q-Q-[Y/H]-Y-R-T-P-P-[T/S] (SEQ ID NO:6)or a sequence that differs from SEQ ID NO:6 by no more than 1 or 2residues; optionally, provided that,

if the light chain variable region segment comprises: a CDR 1 comprisingthe sequence K-S-S-Q-S-V-T-Y-N-Y-K-N-Y-L-A (SEQ ID NO:83); a CDR2comprising the sequence W-A-S-T-R-E-S(SEQ ID NO:84); and a CDR3comprising the sequence Q-Q-Y-Y-R-T-P-P-T (SEQ ID NO:85);

then the heavy chain variable region segment comprises one or more ofthe following: (a) CDRs other than the following: a CDR1 comprising thesequence S-Y-G-M-H (SEQ ID NO:86); a CDR2 comprising the sequenceV-I-S-Y-D-G-S-Y-K-Y-Y-A-D-S-V-Q-G (SEQ ID NO:87); or a CDR3 comprisingthe sequence D-S-E-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:88); or(b) FRs other than the following: an FR1 other thanE-V-Q-L-L-E-S-G-G-G-L-V-K-P-G-Q-S-L-K-L-S-C-A-A-S-G-F-T-F-T (SEQ IDNO:82); an FR2 other than W-V-R-Q-P-P-G-K-G-L-E-W-V-A (SEQ ID NO:75); anFR3 other thanR-F-T-I-S-R-D-N-S-K-N-T-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-A-K (SEQ IDNO:76); or an FR4 other than W-G-A-G-T-T-L-T-V-S-S(SEQ ID NO:89); (c) aCDR1 where the amino residue at position 5 of SEQ ID NO:184 is an S, theamino acid residue at position 6 of SEQ ID NO:184 is a T, or the aminoacid residue at position 8 of SEQ ID NO:184 is an A; (d) a CDR2 whereinthe amino residue at position 2 of SEQ ID NO:2 is a V or an L, the aminoacid at position 4 is an F, the amino acid at position 7 is an N, theamino acid at position 8 is a Y, or the amino acid at position 9 is a R;(e) a CDR3 wherein the amino residue at position 2 of SEQ ID NO:3 is aT, the amino acid residue at position 3 of SEQ ID NO:3 is an R, a K, ora Q, the amino acid residue at position 6 of SEQ ID NO:3 is a T, theamino acid residue at position 15 of SEQ ID NO:3 is an S, the amino acidresidue at position 17 of SEQ ID NO:3 is an L, or a V, the amino acidresidue at position 18 of SEQ ID NO:3 is an L, the amino acid residue atposition 19 of SEQ ID NO:3 is a D, or the amino acid residue at position20 of SEQ ID NO:3 is a Y; (f) an FR1 wherein the amino residue atposition 11 of SEQ ID NO:7 is a Q, or the amino acid residue at position7 of SEQ ID NO:7 is a T; (g) an FR4 wherein the amino residue atposition 3 of SEQ ID NO:10 is a Q, the amino acid residue at position 5of SEQ ID NO:10 is an A; the amino acid residue at position 6 of SEQ IDNO:10 is an M, or the amino acid residue at position 7 of SEQ ID NO:10is a V; or (h) it produces fewer escape mutants than does a referenceanti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114,or CR6261, e.g., when tested by a method disclosed herein, and alsoprovided that, if the heavy chain immunoglobulin variable region segmentcomprises: a CDR1 comprising the sequence S-Y-G-M-H (SEQ ID NO:86); aCDR2 comprising the sequence V-I-S-Y-D-G-S-Y-K-Y-Y-A-D-S-V-Q-G (SEQ IDNO:87); and a CDR3 comprising the sequenceD-S-E-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:88), then the lightchain variable region segment comprises one of more of the following:(a) CDRs other than the following: CDR1 KSSQSVTYNYKNYLA (SEQ ID NO:83);CDR2 WASTRES (SEQ ID NO:84); or CDR3 QQYYRTPPT (SEQ ID NO:85); (b) FRsother than the following: FR1 comprising the sequenceEIVMTQSPDSLAVSLGERATINC (SEQ ID NO:90); FR2 comprising the sequenceWYQQKPGQPPKLLIY (SEQ ID NO:91); FR3 comprising the sequenceGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:92); or

FR4 comprising the sequence FGGGTKLDIK (SEQ ID NO:93); (c) a CDR1wherein the amino residue at position 1 of SEQ ID NO:185 is an R, theamino residue at position 5 of SEQ ID NO:185 is a T, the amino residueat position 6 of SEQ ID NO:185 is an L or an I, the amino residue atposition 7 of SEQ ID NO:185 is an S, the amino residue at position 8 ofSEQ ID NO:185 is an F or a W, or the amino residue at position 9 of SEQID NO:185 is an S or a D; (d) a CDR2 wherein the amino residue atposition 2 of SEQ ID NO:5 is a G, the amino residue at position 4 of SEQID NO:5 is an A, a Y, an H, a K, or a D, the amino residue at position 5of SEQ ID NO:5 is an L, the amino residue at position 7 of SEQ ID NO:5is a T; (e) a CDR3 wherein the amino residue at position 3 of SEQ IDNO:6 is an H; the amino acid residue at position 9 of SEQ ID NO:6 is anS; (f) an FR1 wherein the amino residue at position 1 of SEQ ID NO:11 isa D; the amino residue at position 3 of SEQ ID NO:11 is a Q, the aminoresidue at position 9 of SEQ ID NO:11 is an S, the amino residue atposition 10 of SEQ ID NO:11 is a T, the amino residue at position 11 ofSEQ ID NO:11 is a V, the amino residue at position 12 of SEQ ID NO:11 isan S, the amino residue at position 13 of SEQ ID NO:11 is an A, theamino residue at position 14 of SEQ ID NO:11 is a T, the amino residueat position 15 of SEQ ID NO:11 is a V or an R, the amino residue atposition 17 of SEQ ID NO:11 is a D, the amino residue at position 20 ofSEQ ID NO:11 is an S, the amino residue at position 22 of SEQ ID NO:11is a T, a Q, a D, or an R; (g) an FR2 wherein the amino residue atposition 8 of SEQ ID NO:12 is a K; or the amino residue at position 9 ofSEQ ID NO: 12 is an A; (h) an FR3 wherein the amino residue at position4 of SEQ ID NO: 13 is an E or an S; the amino residue at position 24 ofSEQ ID NO: 13 is a P, the amino residue at position 27 of SEQ ID NO: 13is an F, a K, or a D, the amino residue at position 29 of SEQ ID NO: 13is a T; (i) an FR4 wherein the amino residue at position 3 of SEQ IDNO:14 is a Q, a T, an S, or an N, the amino residue at position 7 of SEQID NO:14 is a V, or the amino residue at position 8 of SEQ ID NO:14 isan E; or (j) it produces fewer escape mutants than does a referenceanti-HA antibody molecule, e.g., Ab 67-11, FI6, FI28, C179, F10, CR9114,or CR6261, e.g., when tested by a method disclosed herein; and furtherprovided that if the light chain variable region segment comprises: aCDR 1 comprising the sequence K-S-S-Q-S-V-T-F-N-Y-K-N-Y-L-A (SEQ IDNO:146); a CDR2 comprising the sequence W-A-S-A-R-E-S(SEQ ID NO:147);and a CDR3 comprising the sequence Q-Q-H-Y-R-T-P-P-T (SEQ ID NO:148);then the heavy chain variable region segment comprises one or more ofthe following: CDRs other than the CDR's described at FIG. 12B; or FRsother than the FRs described at FIG. 12C.

In one embodiment, the heavy chain CDR sequences, collectively, differfrom the recited sequences by no more than 5, 4, 3, 2 or 1 amino acidresidues; and the light chain CDR sequences, collectively, differ fromthe recited sequences by no more than 5, 4, 3, 2 or 1 amino acidresidues.

In one aspect, the disclosure features an isolated nucleic acid moleculecomprising a nucleotide sequence that encodes a heavy chainimmunoglobulin variable region segment featured in the disclosure.

In another aspect, the disclosure features an isolated nucleic acidmolecule that comprises a nucleotide sequence encoding a light chainimmunoglobulin variable region segment featured in the disclosure.

In yet another aspect, the disclosure features an isolated nucleic acidmolecule that comprises a nucleotide sequence that encodes a heavy chainimmunoglobulin variable region segment featured in the disclosure and alight chain immunoglobulin variable region segment featured in thedisclosure.

In yet another aspect, the disclosure features a recombinant vector,such as an expression vector, that comprises a nucleic acid moleculethat comprises a nucleotide sequence that encodes a heavy chainimmunoglobulin variable region segment or nucleotide sequence thatencodes a light chain immunoglobulin variable region segment featured inthe disclosure.

In one aspect, the disclosure features a recombinant vector, such as anexpression vector, that comprises a nucleotide sequence that encodes aheavy chain immunoglobulin variable region segment and a nucleotidesequence that encodes a light chain immunoglobulin variable regionsegment featured in the disclosure.

In one embodiment, the nucleic acid molecules in the recombinant vectorinclude a nucleotide sequence encoding (a) a heavy chain immunoglobulinvariable region segment comprising the amino acid sequence of: S-Y-A-M-H(SEQ ID NO:68) in CDR1; V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G (SEQ ID NO:69)in CDR2; and D-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70) inCDR3; and (b) a light chain immunoglobulin variable region segmentcomprising the amino acid sequence of: Q-S-I-T-F-D-Y-K-N-Y-L-A (SEQ IDNO:145) in CDR1; W-G-S-Y-L-E-S(SEQ ID NO:72) in CDR2; andQ-Q-H-Y-R-T-P-P-S(SEQ ID NO:73) in CDR3.

In one aspect, the disclosure features a cell containing a recombinantvector featured in the disclosure, such as a recombinant vectorcomprising a nucleic acid sequence that encodes a heavy chainimmunoglobulin variable region, or a recombinant vector comprising anucleic acid sequence that encodes a light chain immunoglobulin variableregion. In one embodiment, the cell contains a recombinant vectorcomprising a nucleic acid sequence that encodes a heavy chainimmunoglobulin variable region, and a recombinant vector comprising anucleic acid sequence that encodes a light chain immunoglobulin variableregion. In yet another embodiment, the cell contains a recombinantvector comprising a nucleic acid sequence that encodes a heavy chainimmunoglobulin variable region, and a nucleic acid sequence that encodesa light chain immunoglobulin variable region.

In one aspect, the disclosure features a method of making an antibodymolecule featured in the invention, such as by providing a host cellcomprising a nucleic acid sequence expressing a heavy chain segment anda nucleic acid sequence expressing a light chain segment, and expressingthe nucleic acids in the host cell. In one embodiment, the nucleic acidsequence expressing the heavy chain segment and the nucleic acidsequence expressing the light chain segment are on the same recombinantexpression vector. In another embodiment, the nucleic acid sequenceexpressing the heavy chain segment and the nucleic acid sequenceexpressing the light chain segment are on separate recombinantexpression vectors.

In one aspect, the disclosure features a pharmaceutical compositioncontaining an antibody molecule featured in the disclosure, and apharmaceutically acceptable carrier.

In another aspect, the disclosure features a method of treating orpreventing infection with an influenza virus (e.g., an influenza Avirus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, or aninfluenza B virus, e.g., B/Wisconsin/1/2010), in a subject, e.g., ahuman subject, that comprises:

administering a binding agent, e.g., an antibody molecule, featured inthe disclosure to a subject, e.g., human subject, in need thereof.

In one embodiment, the influenza A virus is an H1, H5, H9, H3 or H7strain, such as an H1N1 strain, an H3N2 strain, or an H5N1 strain ofinfluenza A virus.

In an embodiment the administration results in, or correlates with, oneor more of a reduction in the incidence or severity of a symptom ormanifestation of an influenza infection, or the delay or onset of asymptom or manifestation of an influenza infection.

In an embodiment the administration results in, or correlates with, oneor more of a reduction in the incidence or severity of a symptom ormanifestation of a secondary infection, or the delay or onset of asymptom or manifestation of a secondary infection.

In embodiments the subject, e.g., a human subject, has beenadministered, or the method comprises, administering, or recommendingthe administration of, a second or additional therapy.

In embodiments the antibody molecule is administered in combination witha second or additional agent or therapy.

In embodiments the second or additional therapy comprises administrationof a vaccine or an anti-viral therapy, e.g., an anti-NA or an anti-M2therapy.

In an embodiment the second or additional therapy comprises aadministration of a vaccine, e.g., a vaccine described herein or amixture (a.k.a. a cocktail) of influenza peptides to stimulate thepatient's immune system to prevent infection with particular strains ofinfluenza A.

In an embodiment the second or additional agent comprises administeringan anti-viral agent, a pain reliever, an anti-inflammatory, anantibiotic, a steroidal agent, a second therapeutic antibody molecule(e.g., an anti-HA antibody), an adjuvant, a protease or glycosidase(e.g., sialidase).

In an embodiment the second or additional agent comprises, acyclovir,ribavirin, amantadine, remantidine, a neuraminidase inhibitor (e.g.,zanamivir (Relenza®), oseltamivir (Tamiflu®), laninamivir, peramivir),or rimantadine.

In an embodiment the second or additional agent comprises a secondantibody molecule, e.g., Ab 67-11 (U.S. Provisional application No.61/645,453, FI6 (U.S. Published Application No. 2010/0080813), FI28(U.S. Published Application No. 2010/0080813), C179 (Okuno et al., J.Virol. 67:2552-8, 1993), F10 (Sui et al., Nat. Struct. Mol. Biol.16:265, 2009), CR9114 (Dreyfus et al., Science 337:1343, 2012), orCR6261 (see, e.g., Ekiert et al., Science 324:246, 2009). Thus, Ab 044can be used in combination of any of those antibodies.

In an embodiment the second or additional agent comprises a second oradditional binding agent, e.g., antibody molecule, e.g., an anti-HAantibody, e.g., an disclosed herein. E.g., two or more of Ab 044, Ab069, Ab 032, and Ab 031 can be administered. E.g., Ab 044 can beadministered in combination with Ab 069 or Ab 032

In the case of combinations, two agents can be administered as part ofthe same dosage unit or administered separately. Other exemplary agentsuseful for treating the symptoms associated with influenza infection areacetaminophen, ibuprofen, aspirin, and naproxen.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered to a human subject suffering from or susceptible to aninfluenza infection.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered prior to known exposure to influenza, or to particularinfluenza substypes or strains.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered prior to manifestation of effects or symptoms of influenzainfection, or to one or more particular effects manifestation of effectsor symptoms of influenza infection.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered after known exposure to influenza, or to particularinfluenza substypes or strains.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered after manifestation of effects or symptoms of influenzainfection, or after observation of one or more particular effectsmanifestation of effects or symptoms of influenza infection.

In an embodiment the binding agent, e.g., an antibody molecule, isadministered in response to, or to treat or prevent, a manifestation ofan effect or a symptom of influenza infection, e.g., inflammation,fever, nausea, weight loss, loss of appetite, rapid breathing, increaseheart rate, high blood pressure, body aches, muscle pain, eye pain,fatigue, malaise, dry cough, runny nose, and/or sore throat.

In an embodiment, the method further comprises, testing the humansubject for the influenza virus, e.g., with a method disclosed herein.In embodiments, the administration is responsive to a positive test forinfluenza.

In yet another aspect, the disclosure features a method of treating asubject, e.g., a human subject, infected with an influenza virus (e.g.,an influenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain,e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004, or an influenza B virus, e.g., B/Wisconsin/1/2010)by administering a binding agent, e.g., an antibody molecule, featuredin the disclosure. For example, the influenza A virus is an H1, H5, H9,H3 or H7 strain, such as an H1N1 strain, an H3N2 strain, or an H5N1strain of influenza A virus.

In one embodiment, a binding agent, e.g., an anti-HA antibody, describedherein is administered instead of a vaccine for prevention of influenza.In another embodiment, the binding agent, e.g., anti-HA antibodymolecule, is administered in combination with (simultaneously orsequentially with) a vaccine for prevention of the flu.

In yet another aspect, the disclosure features a method of detectinginfluenza (e.g., influenza A or influenza B) virions in a biologicalsample, such as by contacting the sample with a binding agent, e.g., anantibody molecule, featured in the disclosure, and then detecting thebinding of the antibody molecule to the sample. In one embodiment, themethod of detecting the influenza virus (e.g., influenza A or influenzaB virus) is performed in vitro.

In one aspect, the disclosure features a method of (a) providing asample from a patient; (b) contacting the sample with a binding agent,e.g., an antibody molecule, featured in the disclosure, and (c)determining whether the binding agent, e.g., an antibody molecule,featured in the disclosure binds a polypeptide in the sample, where ifthe binding agent, e.g., an antibody molecule, binds a polypeptide inthe sample, then the patient is determined to be infected with aninfluenza virus (e.g., an influenza A virus, e.g., a Group 1 strain,e.g., an H1N1 strain, e.g., A/South Carolina/1/1918, A/PuertoRico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004, or an influenza B virus,e.g., e.g., B/Wisconsin/1/2010). In one embodiment, the patient isdetermined to be infected with an influenza virus (e.g., an influenza Avirus, e.g., a Group 1 strain, e.g., an H1N1 strain, e.g., A/SouthCarolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009, or anH5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, or aninfluenza B virus, e.g., B/Wisconsin/1/2010), and the patient is furtheradministered a binding agent, e.g., an antibody molecule, disclosedherein, e.g., the binding agent, e.g., an antibody molecule, with whichthe test was performed.

In another aspect, the invention features, a method of inducing immunityto one or more influenza strains, or preventing, delaying or reducinginfection with an influenza strain, or symptom thereof, in a vertebrate,e.g., a human The method comprises administering to the vertebrate,e.g., a human, a broad range vaccine, or broad range immunogen,described herein.

In an embodiment the broad range vaccine, or broad range immunogen,induces an immune response against, or confers protection against, oneor more influenza strains.

In an embodiment the broad range vaccine, or broad range immunogen,induces an immune response against, or confers protection against, twoinfluenza strains.

In an embodiment the broad range vaccine, or broad range immunogen,induces an immune response against, or confers protection against, twoGroup 1 influenza strains.

In an embodiment the broad range vaccine induces, or broad rangeimmunogen, an immune response against, or confers protection against, atleast one Group 1 strain, and a second strain from Group 1, Group2 or aninfluenza B strain.

In one embodiment, the influenza A virus is an H1, H5, H9, H3 or H7strain, such as an H1N1 strain, an H3N2 strain, or an H5N1 strain ofinfluenza A virus.

In an embodiment the administration results in, or correlates with, oneor more of: a reduction in the chance of an infection, a reduction inthe incidence or severity of a symptom or manifestation of an influenzainfection, or the delay or onset of a symptom or manifestation of aninfluenza infection.

In an embodiment the administration results in, or correlates with, oneor more of: a reduction in the incidence or severity of a symptom ormanifestation of a secondary infection, or the delay or onset of asymptom or manifestation of a secondary infection.

In embodiments the subject, e.g., a human subject, has beenadministered, or the method comprises, administering, or recommendingthe administration of, a second or additional therapy.

In embodiments the broad range vaccine is administered in combinationwith a second or additional agent or therapy.

In embodiments the second or additional agent comprises administrationof another vaccine or another anti-viral therapy, e.g., an anti-NA or ananti-M2 therapy.

In an embodiment the second or additional agent comprises administrationof a vaccine comprising a mixture (a.k.a. a cocktail) of influenzapeptides to stimulate the patient's immune system to prevent infectionwith particular strains of influenza A.

In an embodiment the second or additional agent comprises administeringan anti-viral agent, a pain reliever, an anti-inflammatory, anantibiotic, a steroidal agent, a second therapeutic antibody molecule(e.g., an anti-HA antibody), an adjuvant, a protease or glycosidase(e.g., sialidase).

In an embodiment the second or additional agent comprises, acyclovir,ribavirin, amantadine, remantidine, a neuraminidase inhibitor (e.g.,zanamivir (Relenza®), oseltamivir (Tamiflu®), laninamivir, peramivir),or rimantadine.

In an embodiment the second or additional agent comprises an antibodymolecule, e.g., Ab 67-11 (U.S. Provisional application No. 61/645,453,FI6 (U.S. Published Application No. 2010/0080813), FI28 (U.S. PublishedApplication No. 2010/0080813), C179 (Okuno et al., J. Virol. 67:2552-8,1993), F10 (Sui et al., Nat. Struct. Mol. Biol. 16:265, 2009), CR9114(Dreyfus et al., Science 337:1343, 2012), or CR6261 (see, e.g., Ekiertet al., Science 324:246, 2009).

In an embodiment the second or additional agent comprises an antibodymolecule disclosed herein, e.g., an antibody molecule selected fromAb-044, Ab 069, Ab 032, and Ab 031 antibody molecules.

In the case of combinations, two agents can be administered as part ofthe same dosage unit or administered separately.

Other exemplary second or additional agents useful for treating thesymptoms associated with influenza infection are acetaminophen,ibuprofen, aspirin, and naproxen.

In an embodiment the broad range vaccine, or broad range immunogen, isadministered to a human subject suffering from or susceptible to aninfluenza infection.

In an embodiment the broad range vaccine, or broad range immunogen, isadministered prior to known exposure to influenza, or to particularinfluenza substypes or strains.

In an embodiment the broad range vaccine, or broad range immunogen, isadministered prior to manifestation of effects or symptoms of influenzainfection, or to one or more particular effects manifestation of effectsor symptoms of influenza infection.

In an embodiment the broad range vaccine, or broad range immunogen, isadministered after known exposure to influenza, or to particularinfluenza substypes or strains.

In an embodiment the broad range vaccine is administered aftermanifestation of effects or symptoms of influenza infection, or afterobservation of one or more particular effects manifestation of effectsor symptoms of influenza infection.

In an embodiment the broad range vaccine, or broad range immunogen, isadministered in response to, or to treat or prevent, a manifestation ofan effect or a symptom of influenza infection, e.g., inflammation,fever, nausea, weight loss, loss of appetite, rapid breathing, increaseheart rate, high blood pressure, body aches, muscle pain, eye pain,fatigue, malaise, dry cough, runny nose, and/or sore throat.

In an embodiment, the method further comprises, testing the humansubject for the influenza virus, e.g., with a method disclosed herein.In embodiments, the administration is responsive to a positive test forinfluenza.

In yet another aspect, the disclosure features a method of treating asubject, e.g., a human subject, infected with an influenza virus (e.g.,an influenza A virus, e.g., a Group 1 strain, e.g., an H1N1 strain,e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004, or an influenza B virus, e.g., B/Wisconsin/1/2010)by administering a broad range vaccine featured in the disclosure. Forexample, the influenza A virus is an H1, H5, H9, H3 or H7 strain, suchas an H1N1 strain, an H3N2 strain, or an H5N1 strain of influenza Avirus.

In another aspect, the invention features, a method of reducing theseverity of influenza in a population. The method includes administeringa broad range vaccine, or broad range immunogen, to sufficientindividuals in the population to prevent or decrease the chance ofinfluenza virus transmission to another individual in the population.

In another aspect, the invention features, a kit comprising one or morecontainers having disposed therein a broad range immunogen, a nucleicacid encoding the broad range epitope, or a broad range vaccinedescribed herein. In an embodiment the kit includes a container havingadjuvant disposed therein. In an embodiment the kit comprises a deliverydevice, e.g., an injection device or inhaler. In an embodiment the broadrange epitope described herein, or a nucleic acid encoding the broadrange epitope or broad reange vaccine, is disposed in a delivery device.

In an embodiment the kit comprises a delivery device, e.g., an injectiondevice or inhaler. In an embodiment the vaccine is disposed in adelivery device.

In another aspect, the invention features, a composition, e.g., avaccine, comprising a broad range epitope described herein, packaged ina hermetically sealed container such as an ampoule. In an embodiment,the composition a liquid. In an embodiment, the composition is liquid adry sterilized lyophilized powder or water free concentrate.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

The details of one or more embodiments featured in the disclosure areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages featured in the disclosure will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the heavy and light chain amino acid sequences (SEQ ID NOs:94and 95, respectively) of the anti-HA antibody A18. The constant domainsequence is indicated by italics. The CDRs are indicated by underlining

FIG. 2 is the variable heavy chain domain sequence of exemplary anti-HAantibodies. The SEQ ID NOs. for sequences shown are as follows: VH15 isSEQ ID NO: 15; VH16 is SEQ ID NO: 16; VH17 is SEQ ID NO: 17; VH18 is SEQID NO: 18; VH19 is SEQ ID NO: 19; VH21 is SEQ ID NO: 21; VH22 is SEQ IDNO: 22; VH20 is SEQ ID NO: 20; VH23 is SEQ ID NO: 23; VH24 is SEQ ID NO:24; VH25 is SEQ ID NO: 25; VH26 is SEQ ID NO: 26; VH27 is SEQ ID NO: 27;and VH161 is SEQ ID NO: 161.

FIG. 3 is the variable light chain domain sequence of exemplary anti-HAantibodies. The SEQ ID NOs. for sequences shown are as follows: VL28 isSEQ ID NO: 28; VL29 is SEQ ID NO: 29; VL30 is SEQ ID NO: 30; VL35 is SEQID NO: 35; VL31 is SEQ ID NO: 31; VL32 is SEQ ID NO: 32; VL33 is SEQ IDNO: 33; VL34-ID is SEQ ID NO: 34; VL36 is SEQ ID NO: 36; VL45 is SEQ IDNO: 45; VL46 is SEQ ID NO: 46; VL37 is SEQ ID NO: 37; VL38 is SEQ ID NO:38; VL39 is SEQ ID NO: 39; VL40 is SEQ ID NO: 40; VL41 is SEQ ID NO: 41;VL42 is SEQ ID NO: 42; VL43 is SEQ ID NO: 43; VL44 is SEQ ID NO: 44;VL47 is SEQ ID NO: 47; VL48 is SEQ ID NO: 48; VL49 is SEQ ID NO: 49;VL50 is SEQ ID NO: 50; VLSI is SEQ ID NO: 51; VL52 is SEQ ID NO: 52;VL53 is SEQ ID NO: 53; VL54 is SEQ ID NO: 54; VL55 is SEQ ID NO: 55;VL56 is SEQ ID NO: 56; VL57 is SEQ ID NO: 57; VL58 is SEQ ID NO: 58;VL59 is SEQ ID NO: 59; VL60 is SEQ ID NO: 60; VL61 is SEQ ID NO: 61;VL153 is SEQ ID NO: 153; VL154 is SEQ ID NO: 154; VL155 is SEQ ID NO:155; VL156 is SEQ ID NO: 156; and VL62 is SEQ ID NO: 62.

FIG. 4 is a graph depicting neutralization of the H3N2 strain X-31 byA18 antibodies. In contrast, strain X-31 was not neutralized by AB1antibodies.

FIG. 5 is a graph of ELISA results depicting stronger binding of A18 toH3 Brisbane/07, and much weaker binding to H3 Wyoming/03.

FIG. 6 is a graph of ELISA results depicting binding of A18 to H3 Bris07HA, H3N2 Bris07 virus, and weak binding to the negative control, BSA(bovine serum albumin)

FIGS. 7A and 7B are graphs depicting the ability of the A18 antibody toprotect infected mice from weight loss. The mice were infected eitherwith H1N1 (FIG. 7A) or with H3N2 (FIG. 7B) influenza strains.

FIGS. 8A and 8B are graphs depicting percent survival of mice infectedwith H1N1 (FIG. 8A) or H3N2 (FIG. 8B), and treated 48 hours later witheither Ab 028, Ab 031, or Ab 032 antibodies.

FIGS. 9A, 9B and 9C are graphs depicting percent survival of miceinfected with H1N1 (A/Puerto Rico/8/34) (FIG. 9A), H3N2(A/Victoria/03/75) (FIG. 9B) or H1N1 (A/California/04/09) (FIG. 9C).Mice were treated with various dosages of Ab 032 antibody.

FIGS. 10A to 10D are graphs of ELISA results depicting the broadspecificity of antibodies Ab 014 and Ab 028. Both antibodies are shownto bind to influenza subtypes from Group 1 (subtypes H1, H5, and H9) andfrom Group 2 (subtypes H3 and H7).

FIG. 11 is a graph depicting the inability of the influenza strain PR8(H1N1) to overcome neutralization by AB1 after 5 rounds (R5) ofpropagation in the presence of AB 1. On the other hand, PR8 does developresistance to the control neutralizing antibody C179 seen by titerrecovery after 4 round of selection in the presence of C179.

FIG. 12 shows the amino acid sequences of the heavy chain variableregions of FI6 (SEQ ID NO: 175), FI370 (SEQ ID NO: 176), FI6 variant 1(SEQ ID NO: 177), FI6 variant 3 (SEQ ID NO: 178), FI6/370 (SEQ ID NO:179) and the amino acid sequence of kappa light chain variable region ofFI6 (SEQ ID NO: 180).

FIG. 13 is the variable heavy chain domain sequence of exemplary anti-HAantibodies as shown in FIG. 2 and including an N-terminal ID dipeptide.The SEQ ID NOs. for sequences shown are as follows: VH15-ID is SEQ IDNO: 96; VH16-ID is SEQ ID NO: 97; VH17-ID is SEQ ID NO: 98; VH18-ID isSEQ ID NO: 99; VH19-ID is SEQ ID NO: 100; VH21-ID is SEQ ID NO: 101;VH22-ID is SEQ ID NO: 102; VH20-ID is SEQ ID NO: 103; VH23-ID is SEQ IDNO: 104; VH24-ID is SEQ ID NO: 105; VH25-ID is SEQ ID NO: 106; VH26-IDis SEQ ID NO: 107; VH27-ID is SEQ ID NO: 108; and VH161-ID is SEQ ID NO:109.

FIG. 14 is the variable light chain domain sequence of exemplary anti-HAantibodies as shown in FIG. 3 and including an N-terminal ID dipeptide.The SEQ ID NOs. for sequences shown are as follows: VL28-ID is SEQ IDNO: 110; VL29-ID is

SEQ ID NO: 111; VL30-ID is SEQ ID NO: 112; VL35-ID is SEQ ID NO: 113;VL31-ID is SEQ ID NO: 114; VL32-ID is SEQ ID NO: 115; VL33-ID is SEQ IDNO: 116; VL34-ID is SEQ ID NO: 117; VL36-ID is SEQ ID NO: 118; VL45-IDis SEQ ID NO: 119; VL46-ID is SEQ ID NO: 120; VL37-ID is SEQ ID NO: 121;VL38-ID is SEQ ID NO: 122; VL39-ID is SEQ ID NO: 123; VL40-ID is SEQ IDNO: 124; VL41-ID is SEQ ID NO: 125; VL42-ID is SEQ ID NO: 126; VL43-IDis SEQ ID NO: 127; VL44-ID is SEQ ID NO: 128; VL47-ID is SEQ ID NO: 129;VL48-ID is SEQ ID NO: 130; VL49-ID is SEQ ID NO: 131; VL50-ID is SEQ IDNO: 132; VL51-ID is SEQ ID NO: 133; VL52-ID is SEQ ID NO: 134; VL53-IDis SEQ ID NO: 135; VL54-ID is SEQ ID NO: 136; VL55-ID is SEQ ID NO: 137;

VL56-ID is SEQ ID NO: 138; VL57-ID is SEQ ID NO: 139; VL58-ID is SEQ IDNO: 140; VL59-ID is SEQ ID NO: 141; VL60-ID is SEQ ID NO: 142; VL61-IDis SEQ ID NO: 143; VL153-ID is SEQ ID NO: 157; VL154-ID is SEQ ID NO:158; VL155-ID is SEQ ID NO: 159; VL156-ID is SEQ ID NO: 160; and VL62-IDis SEQ ID NO: 144.

FIG. 15 is a graph depicting the number of syncytia observed aftertreatment of HA infected HEK293 cells with Ab 032 antibody. “Pre” and“Post” indicates treatment of the cells with antibody before inductionof fusion conditions by low pH (5.0), or after induction by low pH.

FIG. 16 is a graph depicting percent survival of mice infected with H1N1and subsequently infected with the bacteria S. pneumococcus. Mice weretreated with antibiotic or anti-HA antibody.

FIG. 17 shows the variable light and heavy chain sequences of additionalexemplary anti-HA antibodies. The SEQ ID NOs. for sequences shown are asfollows: VL165 is SEQ ID NO: 165; VL166 is SEQ ID NO: 166; VL167 is SEQID NO: 167; VL168 is SEQ ID NO: 168; VL169 is SEQ ID NO: 169; VH164 isSEQ ID NO: 164; VH162 is SEQ ID NO: 162; VH163 is SEQ ID NO: 163.

FIG. 18 is a graph depicting the number of syncytia observed aftertreatment of HA infected HEK293 cells with Ab 044 antibody. Left bar=pH5.0; right bar=pH 7.0.

FIG. 19A shows the weight changes of mice after challenge with H1N1strain PR8 and administration of vehicle, ribavirin, Ab 044 prophylaxisat 10 mg/kg, Ab 044 prophylaxis at 2.5 mg/kg, or Ab 044 prophylaxis at0.6 mg/kg.

FIG. 19B shows the weight change of mice after challenge with H1N1strain PR8 and administration of Ab 044 therapy two days post infectionat 10 mg/kg, 2.5 mg/kg, or 0.6 mg/kg; or Ab 044 therapy three days postinfection at 20 mg/kg.

FIG. 20A shows the weight changes of mice after challenge with H3N2strain Victoria and administration of vehicle, ribavirin, or AB 044prophylaxis at 10 mg/kg.

FIG. 20B shows the weight changes of mice after challenge with H3N2 andadministration of Ab 044 therapy two days post infection at 10 mg/kg,2.5 mg/kg, or 0.6 mg/kg; or Ab 044 therapy three days post infection at20 mg/kg.

FIG. 21A shows lung viral load in mice four days after viral challengewith H1N1, as measured by plaque assay. Infected mice were treated withvehicle, ribavirin,

Ab 044 prophylaxis at 10 mg/kg, Ab 044 prophylaxis at 2.5 mg/kg, Ab 044prophylaxis at 0.6 mg/kg, Ab 044 therapy at 10 mg/kg, Ab 044 therapy at2.5 mg/kg, Ab 044 therapy at 0.6 mg/kg, or Ab 044 therapy at 72 h.

FIG. 21B shows lung viral load in mice four days after viral challengewith H3N2. Infected mice were treated with vehicle, ribavirin, Ab 044prophylaxis at 10 mg/kg, Ab 044 therapy at 10 mg/kg, Ab 044 therapy at2.5 mg/kg, Ab 044 therapy at 0.6 mg/kg, or Ab 044 therapy at 72 h.Asterisk, p<0.05.

FIG. 22A shows the survival curves of mice after H1N1 challenge andadministration of vehicle, ribavirin, Ab 044 prophylaxis at 10 mg/kg,2.5 mg/kg, or 0.6 mg/kg.

FIG. 22B shows the survival curves of mice after H1N1 challenge andadministration of vehicle, Ab 044 therapy at 48 h at 10 mg/kg, at 2.5mg/kg, at 0.6 mg/kg, or Ab 044 therapy at 72 h at 20 mg/kg.

FIG. 23A shows the survival curves of mice after H3N2 challenge andadministration of vehicle, ribavirin, or Ab 044 prophylaxis at 10 mg/kg.

FIG. 23B shows the survival curves of mice after H3N2 challenge andadministration of vehicle, Ab 044 therapy at 48 h post infection at 10mg/kg, at 2.5 mg/kg, at 0.6 mg/kg, or Ab 044 therapy at 72 h postinfection at 20 mg/kg.

FIG. 24 depicts the effects of prophylactic and therapeuticadministration of Ab 044 on the weight of BALB/c mice infected withinfluenza A/Vietnam/1203/2004 H5N1 virus.

FIG. 25 depicts the effects of prophylactic and therapeuticadministration of Ab 044 on survival of BALB/c mice infected withinfluenza A/Vietnam/1203/2004 H5N1 virus.

FIG. 26 is a three dimensional representation of H3 HA with the aminoacids residues that are predicted to be part of Ab044 epitope but notpart of FI6's epitope highlighted. That is, the highlighted amino acidsare unique to Ab044's epitope.

FIG. 27 is a three dimensional representation of H3 HA with the aminoacid residues that are part of FI6's epitope but not predicted to bepart of Ab044's epitope highlighted.

DETAILED DESCRIPTION

The disclosure is based, at least in part, on the design and synthesisof antibody molecules that can bind an epitope that is conserved acrossmultiple hemagglutinin subtypes of influenza viruses (e.g., influenza Aand influenza B viruses). For example, the antibody molecules describedherein are useful as broad spectrum therapy against disease caused by atleast one influenza A strain belonging to Group 1 and one influenza Astrain belonging to Group 2 to neutralize infectivity of virusesbelonging to both Group 1 and Group 2 (at least one subtype of each).

The antibody molecules were designed by a rational structure-basedapproach to target a region on the virus that is not fully accessible tothe human immune system and, therefore, not amenable to antibodyselection through more classical screening approaches. Thisrational-based approach to the design and development of broad-spectrumantibody molecules allows for the development of more efficaciousvaccines for pandemic and seasonal influenza. This approach also allowsfor the advance preparation of pandemic vaccines so that they are readyto be employed against specific virus subtypes (e.g., avian virussubtypes) that may mutate to become human-adapted and highlytransmissible. Vaccines (e.g., seasonal vaccines) that utilize theantibody molecules described herein can generate a more potent immuneresponse without the use of adjuvants and provide broad protectionagainst viral strain variation.

Definitions

As used herein, the term “antibody molecule” refers to a polypeptidethat comprises sufficient sequence from an immunoglobulin heavy chainvariable region and/or sufficient sequence from an immunoglobulin lightchain variable region, to provide antigen specific binding. It comprisesfull length antibodies as well as fragments thereof, e.g., Fabfragments, that support antigen binding. Typically an antibody moleculewill comprise heavy chain CDR1, CDR2, and CDR3 and light chain CDR1,CDR2, and CDR3 sequence. Antibody molecules include human, humanized,CDR-grafted antibodies and antigen binding fragments thereof. Inembodiments an antibody molecule comprises a protein that comprises atleast one immunoglobulin variable region segment, e.g., an amino acidsequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence.

The VH or VL chain of the antibody molecule can further include all orpart of a heavy or light chain constant region, to thereby form a heavyor light immunoglobulin chain, respectively. In one embodiment, theantibody molecule is a tetramer of two heavy immunoglobulin chains andtwo light immunoglobulin chains.

An antibody molecule can comprise one or both of a heavy (or light)chain immunoglobulin variable region segment. As used herein, the term“heavy (or light) chain immunoglobulin variable region segment,” refersto an entire heavy (or light) chain immunoglobulin variable region, or afragment thereof, that is capable of binding antigen. The ability of aheavy or light chain segment to bind antigen is measured with thesegment paired with a light or heavy chain, respectively. In someembodiment, a heavy or light chain segment that is less than a fulllength variable region will, when paired with the appropriate chain,bind with an affinity that is at least 20, 30, 40, 50, 60, 70, 80, 90,or 95% of what is seen when the full length chain is paired with a lightchain or heavy chain, respectively.

An immunoglobulin variable region segment may differ from a reference orconsensus sequence. As used herein, to “differ,” means that a residue inthe reference sequence or consensus sequence is replaced with either adifferent residue or an absent or inserted residue.

An antibody molecule can comprise a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody comprisestwo heavy (H) chain variable regions and two light (L) chain variableregions or antibody binding fragments thereof. The light chains of theimmunoglobulin may be of types kappa or lambda. In one embodiment, theantibody molecule is glycosylated. An antibody molecule can befunctional for antibody dependent cytotoxicity and/orcomplement-mediated cytotoxicity, or may be non-functional for one orboth of these activities. An antibody molecule can be an intact antibodyor an antigen-binding fragment thereof.

Antibody molecules include “antigen-binding fragments” of a full lengthantibody, e.g., one or more fragments of a full-length antibody thatretain the ability to specifically bind to an HA target of interest.Examples of binding fragments encompassed within the term“antigen-binding fragment” of a full length antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′) or F(ab′)₂ fragment, a bivalent fragmentincluding two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR) that retains functionality. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules knownas single chain Fv (scFv). See e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883. Antibody molecules include diabodies.

As used herein, an antibody refers to a polypeptide, e.g., a tetramericor single chain polypeptide, comprising the structural and functionalcharacteristics, particularly the antigen binding characteristics, of animmunoglobulin. Typically, a human antibody comprises two identicallight chains and two identical heavy chains. Each chain comprises avariable region.

The variable heavy (VH) and variable light (VL) regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). Human antibodies have threeVH CDRs and three VL CDRs, separated by framework regions FR1-FR4. Theextent of the FRs and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).Kabat definitions are used herein. Each VH and VL is typically composedof three CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, 1-R4.

The heavy and light immunoglobulin chains can be connected by disulfidebonds. The heavy chain constant region typically comprises threeconstant domains, CH1, CH2 and CH3. The light chain constant regiontypically comprises a CL domain. The variable region of the heavy andlight chains contains a binding domain that interacts with an antigen.The constant regions of the antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells of theimmune system (e.g., effector cells) and the first component (C1q) ofthe classical complement system.

The term “immunoglobulin” comprises various broad classes ofpolypeptides that can be distinguished biochemically. Those skilled inthe art will appreciate that heavy chains are classified as gamma, mu,alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them(e.g., γ1-γ4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are clearly within the scope ofthe present disclosure. Light chains are classified as either kappa orlambda (κ, λ). Each heavy chain class may be bound with either a kappaor lambda light chain.

Suitable antibodies include, but are not limited to, monoclonal,monospecific, polyclonal, polyspecific, human antibodies, primatizedantibodies, chimeric antibodies, bi-specific antibodies, humanizedantibodies, conjugated antibodies (i.e., antibodies conjugated or fusedto other proteins, radiolabels, cytotoxins), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain antibodies, cameloidantibodies, and antibody fragments.

In embodiments, an antibody is a humanized antibody. A humanizedantibody refers to an immunoglobulin comprising a human framework regionand one or more CDR's from a non-human, e.g., mouse or rat,immunoglobulin. The immunoglobulin providing the CDR's is often referredto as the “donor” and the human immunoglobulin providing the frameworkoften called the “acceptor,” though in embodiments, no source or noprocess limitation is implied. Typically a humanized antibody comprisesa humanized light chain and a humanized heavy chain immunoglobulin.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules Immunoglobulin domainstypically contain two β-sheets formed of about seven β-strands, and aconserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay(1988) Ann. Rev. Immunol. 6:381-405).

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence that can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that comprises an immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that interacts with the target antigen.

As used herein, the term antibodies comprises intact monoclonalantibodies, polyclonal antibodies, single domain antibodies (e.g., sharksingle domain antibodies (e.g., IgNAR or fragments thereof)),multispecific antibodies (e.g., bi-specific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity. Antibodies for use herein maybe of any type (e.g., IgA, IgD, IgE, IgG, IgM).

The antibody or antibody molecule can be derived from a mammal, e.g., arodent, e.g., a mouse or rat, horse, pig, or goat. In embodiments, anantibody or antibody molecule is produced using a recombinant cell. Insome embodiments an antibody or antibody molecule is a chimericantibody, for example, from mouse, rat, horse, pig, or other species,bearing human constant and/or variable regions domains.

A binding agent, as used herein, is an agent that bind, e.g.,specifically binds, a target antigen, e.g., HA. Binding agents of theinvention share sufficient structural relationship with anti-HA antibodymolecules disclosed herein to support specific binding to HA, and inembodiments, other functional properties of an anti-HA antibody moleculedisclosed herein. In embodiments a binding agent will exhibit a bindingaffinity at of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of anantibody molecule disclosed herein, e.g., an antibody molecule withwhich it shares, significant structural homology, e.g., CDR sequences.Binding agents can be naturally occurring, e.g., as are some antibodies,or synthetic. In an embodiment a binding agents is a polypeptide, e.g.,an antibody molecule, e.g., an antibody. While some binding agents areantibody molecules, other molecules, e.g., other polypeptides, can alsofunction as binding agents. Polypeptide binding agents can be monomericor multimeric, e.g., dimeric, trimeric, or tetrameric and can bestabilized by intra- or interchain bonds, e.g., disulfide bonds. Theycan contain natural or non-naturally occurring amino acid residues. Inembodiments, binding agents are antibody molecules, or otherpolypeptides, that present one or more CDRs of antibody moleculesdisclosed herein or that otherwise mimic the structure of an antibodymolecule disclosed herein. Binding agents can also comprise aptomers,nucleic acids or other molecular entities. A binding agent can bedeveloped in a variety of ways, e.g., by innumization, by rationaldesign, screening of random structures, or a combination of those orother approaches. Typically a binding agent will act by making contactwith substantially the same epitope as an antibody molecule disclosedherein, e.g., an antibody molecule with which it shares, significantstructural homology, e.g., CDR sequences. A binding agent can interactwith amino acids, saccharides, or combinations thereof. Polypeptidesother than antibodies can be used as a scaffold to present sequence,e.g., one or more, or a complete set of heavy chain and/or light chainCDRs, disclosed herein. Exemplary scaffolds include adnectin, zincfinger DNA-binding proteins. protein A, lipoclins, ankryin consensusrepeat domain, thioredoxin, anticalins, centyrin, avimer domains,ubiquitin, peptidomimetics, stapled peptides, cystine-knot miniproteins,and IgNARs. In some embodiments, a binding agent is or comprises anucleic acid, e.g., DNA, RNA or mixtures thereof. In embodiments abinding agent, e.g., a nucleic acid, shows secondary, tertiary, orquaternary structure. In some embodiments a binding agent, e.g., anucleic acid, forms a structure that mimics the structure of an antibodymolecule disclosed herein.

A broad spectrum binding agent, e.g., antibody molecule, as used herein,binds, a plurality of different HA molecules, and optionally neutralizesviruses comprising the different HA molecules. In an embodiment it bindsa first HA and binds a second HA from influenza A Group 1, andoptionally neutralizes viruses comprising the first or second HAmolecules. In an embodiments it binds a first HA from an influenza AGroup 1 virus, and binds a second HA from an influenza A Group 2 virus,and optionally neutralizes viruses comprising the different HAmolecules. In an embodiment it binds a first HA from an influenza AGroup 1 or 2 virus and binds a HA from an influenza B virus, andoptionally neutralizes viruses comprising the different HA molecules. Inan embodiments it binds, and in embodiments neutralizes, at least twodifferent clades or clusters of virus, e.g., from different Groups. Inembodiments it binds, and in embodiments neutralizes, all orsubstantially all strains of Group 1 an/or Group 2 disclosed herein. Inan embodiment, a binding agent, e.g., antibody molecule, binds, and inembodiments, neutralizes: at least one strain from the Group 1 H1, e.g.,H1a or H1b, cluster and at least one strain from the Group 2 H3 or H7cluster. In an embodiment, a binding agent, e.g., antibody molecule,binds, and in embodiments, neutralizes: at least one strain from theGroup 1 H1, e.g., H1a or H1b, cluster and at least one influenza Bstrain. In an embodiment, a binding agent, e.g., antibody molecule,binds, and in embodiments, neutralizes: at least one strain from theGroup 2 H3 or H7 cluster and at least one influenza B strain. In anembodiment, a binding agent, e.g., antibody molecule, binds, and inembodiments, neutralizes: at least one strain from the Group 1 H1, e.g.,H1a or H1b, cluster, at least one strain from the Group 2 H3 or H7cluster, and at least one influenza B strain. In some embodiments,binding agent, e.g., antibody molecule, binds, and optionallyneutralizes or mediate infection of particular hosts, e.g., avian,camel, canine, cat, civet, equine, human, mouse, swine, tiger, or othermammal or bird.

The term “combination therapy”, as used herein, refers to administrationof a plurality of agents, e.g., wherein at least one binding agent,e.g., antibody molecule, disclosed herein is administered to a subject,e.g., a human subject. The introduction of the agents into the subjectcan be at different times. In embodiments the agents are administered inoverlapping regimens, or such that the subject is simultaneously exposedto both agents, or such that the response of the subject is better thanwould be seen with either agent administered alone.

As used herein, an “escape mutant” is a mutated influenza strain that isresistant to neutralization by an anti-HA antibody molecule describedherein. In embodiments an escape mutant is resistant to neutralizationwith a binding agent, e.g., antibody molecule, but its parent strain isneutralized by the binding agent, e.g., antibody molecule.

As used herein, “pandemic influenza” refers to a new viral strain thatarises due to human adaptation of an influenza strain by mutation or byemergence of a strain by reassortment of different strains of influenzaA. The resulting pandemic strain is significantly different fromprevious strains and most people will have little or no pre-existingimmunity. Symptoms and complications may be more severe and morefrequent than those typical of seasonal influenza. Examples of pastpandemic flu viruses include, e.g., the 2009 H1N1 ‘swine flu,’ the1957-58 H2N2 ‘Asian flu’ and the 1968 H3N2 influenza strains.

The terms “purified” and “isolated” as used herein in the context of anantibody molecule, e.g., a antibody, a immunogen, or generally apolypeptide, obtained from a natural source, refers to a molecule whichis substantially free of contaminating materials from the naturalsource, e.g., cellular materials from the natural source, e.g., celldebris, membranes, organelles, the bulk of the nucleic acids, orproteins, present in cells. Thus, a polypeptide, e.g., an antibodymolecule, that is isolated includes preparations of a polypeptide havingless than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellularmaterials and/or contaminating materials. The terms “purified” and“isolated” when used in the context of a chemically synthesized species,e.g., an antibody molecule, or immunogen, refers to the species which issubstantially free of chemical precursors or other chemicals which areinvolved in the syntheses of the molecule.

A preparation of binding agents, e.g., antibody molecules, as usedherein, comprises a plurality of molecules of a binding agent, e.g.,antibody molecule, described herein. In embodiments that binding agent,e.g., antibody molecule, makes up at least 60, 70, 80, 90, 95, 98, 99,99.5 or 99.9%, of the preparation, or of the active ingredients of thepreparation, by weight or number. In embodiments that binding agent isan antibody molecule which makes up at least 60, 70, 80, 90, 95, 98, 99,99.5 or 99.9%, of the preparation, or of the active ingredients, orpolypeptide ingredients, or antibody molecules, of the preparation, byweight or number. In embodiments the binding agent is an antibodymolecule and the preparation contains no more than 30, 20, 10, 5, 2, 1,or 0.5%, by weight or number, of a contaminant, e.g., a reactant,solvent, precursor or other species, from the source, or used in thepreparation, of the antibody molecule, e.g., a species from a cell,reaction mixture, or other system used to produce the antibody molecule.

As used herein, the term “prevent infection” means that a subject (e.g.,a human) is less likely to be infected by influenza if the subjectreceives the antibody prior to (e.g., 1 day, 2 days, 1 week, 2 weeks, 3weeks, or 1 month of more) before being exposed to influenza.

As used herein, “seasonal influenza” is a strain that is identical orclosely related to strains that have been circulating in the humanpopulation in recent years and therefore most people are at leastpartially immune to it. Such a strain is not likely to cause severedisease. Symptoms can include fever, cough, runny nose, and muscle pain,and in rare cases, death can result from complications, such aspneumonia. Outbreaks follow predictable seasonal patterns, annually, andusually in fall and winter and in temperate climates. Infection due toseasonal influenza is commonly referred to as the flu.

As used herein, specific binding, means that a binding agent, e.g., anantibody molecule, binds its antigen with a K_(D) of equal to or lessthan 10⁻⁵. In embodiments, the antibody binds it's antigen with a K_(D)of equal to or less than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹².

As used herein, the term “therapeutically effective amount” refers to anamount of a therapeutic agent, e.g., a binding agent, e.g., an antibodymolecule, which results in a positive outcome for the subject. Inembodiments, it can be statistically correlated with therapeutic effector benefit, e.g., the lessening or prevention of a manifestation of aneffect or a symptom, when administered to a population of subjects. Inembodiments it is an amount that also provides a preselected, orreasonable, benefit/risk ratio. In embodiments it is an amount effectiveto reduce the incidence and/or severity of and/or to delay onset of oneor more features, symptoms, or characteristics of a disease, disorder,or condition. A therapeutically effective amount is can be administeredin a dosing regimen that may comprise one or multiple unit doses.

As used herein, the term “treat infection” means that a subject (e.g., ahuman) who has been infected with an influenza and experiences symptomsof the influenza (e.g., the flu), will in embodiments, suffer lesssevere symptoms and/or will recover faster when the antibody molecule isadministered than if the antibody is never administered. In embodiments,when an infection is treated, an assay to detect virus in the subjectwill detect less virus after effective treatment for the infection. Forexample, a diagnostic assay using an antibody molecule, such as anantibody molecule described herein, will detect less or no virus in abiological sample of a patient after administration of an antibodymolecule for the effective treatment of the viral infection. Otherassays, such as PCR (e.g., qPCR) can also be used to monitor treatmentin a patient, to detect the presence, e.g., decreased presence (orabsence) after treatment of viral infection in the patient. Treatmentcan, e.g., partially or completely alleviate, ameliorate, relive,inhibit, reduce the severity of, and/or reduces incidence andoptionally, delay onset of, one or more manifestations of the effects orsymptoms, features, and/or causes of a particular disease, disorder,and/or condition (e.g., influenza. In embodiments treatment is of asubject who does not exhibit signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. In embodiments treatment is ofa subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In embodiments, treatment is of asubject diagnosed as suffering from influenza.

Calculations of “homology” or “sequence identity” or “identity” betweentwo sequences (the terms are used interchangeably herein) can beperformed as follows. The sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The optimal alignment is determined as the best score using the GAPprogram in the GCG software package with a Blossum 62 scoring matrixwith a gap penalty of 12, a gap extend penalty of 4, and a frameshiftgap penalty of 5. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences.

Hemagglutinin (HA) Polypeptides and Influenza

Influenza viruses are negative sense, single-stranded, segmented RNAenvelope viruses. Two glycoproteins, a hemagglutinin (HA) polypeptideand a neuraminidase (NA) polypeptide, are displayed on the outer surfaceof the viral envelope. There are several Influenza A subtypes, labeledaccording to an H number (for the type of hemagglutinin) and an N number(for the type of neuraminidase). There are 17 different H antigens (H1to H17) and nine different N antigens (N1 to N9). Influenza strains areidentified by a nomenclature based on the number of the strain's HApolypeptide and NA polypeptide subtypes, for example, H1N1, H1N2, H1N3,H1N4, H1N5, and the like.

HA is the major viral surface glycoprotein that mediates binding andentry of the virus into host cells and is a primary target ofneutralizing antibody responses. HA is a trimer of three identicalmonomers. Each monomer is synthesized as a precursor, HA₀, that isproteolytically processed into two disulfide-bonded polypeptide chains,HA₁ and HA₂. The ectodomain of this protein has (i) a globular headdomain possessing receptor binding activity and major antigenicdeterminants, (ii) a hinge region, and (iii) a stem region where asequence critical for fusion, the fusion peptide, is located. The viralreplication cycle is initiated when the virion attaches via its surfacehemagglutinin proteins to sialylated glycan receptors on the host celland enters the cell by endocytosis. The acidic environment in theendosome induces conformational changes in HA that expose the fusionpeptide hidden within the stem region of the trimer. The exposed fusionpeptide mediates the fusion of the viral and target cell membranesresulting in the release of the viral ribonucleoprotein into the cellcytoplasm.

Influenza A hemagglutinin subtypes have been divided into two maingroups and four smaller clades, and these are further divided intoclusters. Group 1 influenza A strains are divided into 3 clades: (i) H8,H9 and H12 (“the H9 cluster”); (ii) H1, H2, H5, H6 and H17 (“the H1acluster”); and (iii) H11, H13 and H16 (“the H1b cluster”). Group 2strains are divided into 2 clades: (i) H3, H4 and H14 (“the H3cluster”); and (ii) H7, H10 and H15 (“the H7 cluster”). The H1b and theH1a clusters are classified together as the H1 cluster. The different HAsubtypes do not necessarily share strong amino acid sequence identity,but their overall 3D structures are similar.

Of the 17 HA polypeptide subtypes, only 3 (H1, H2 and H3) have adaptedfor human infection. These subtypes have in common an ability to bindalpha 2,6 sialylated glycans. In contrast, their avian counterpartspreferentially bind to alpha 2,3 sialylated glycans. HA polypeptidesthat have adapted to infect humans (e.g., of HA polypeptides from thepandemic H1N1 (1918) and H3N2 (1967-68) influenza subtypes) have beencharacterized by an ability to preferentially bind to α2,6 sialylatedglycans in comparison with their avian progenitors that preferentiallybind to α2,3 sialylated glycans (see, e.g., Skehel & Wiley, Annu RevBiochem, 69:531, 2000; Rogers, & Paulson, Virology, 127:361, 1983;Rogers et al., Nature, 304:76, 1983; Sauter et al., Biochemistry,31:9609, 1992

Further, HA polypeptides that mediate infection of humans preferentiallybind to umbrella topology glycans over cone topology glycans (see, e.g.,U.S. 2011/0201547). Without wishing to be bound by any particulartheory, it has been proposed that the ability to infect human hostscorrelates less with binding to glycans of a particular linkage, andmore with binding to glycans of a particular topology, even thoughcone-topology glycans may be α2,6 sialylated glycans. In has beendemonstrated that HA polypeptides that mediate infection of humans bindto umbrella topology glycans, often showing preference for umbrellatopology glycans over cone topology glycans (See, for example, U.S. Ser.No. 12/348,266 filed Jan. 2, 2009, U.S. Ser. No. 12/301,126, filed Nov.17, 2008, U.S. Ser. No. 61/018,783, filed Jan. 3, 2008, U.S. Ser. No.11/969,040, filed Jan. 3, 2008, U.S. Ser. No. 11/893,171, filed Aug. 14,2007, U.S. Ser. No. 60/837,868, filed on Aug. 14, 2006, U.S. Ser. No.60/837,869, filed on August 14, and to PCT application PCT/US07/18160,filed Aug. 14, 2007.

Mature HA polypeptides include three domains, (i) a globular domain(a.k.a., the head domain) consists mainly of the HA1 peptide andcontains the receptor (sialylated glycoproteins)-binding region, (ii) astalk domain (HA1 and HA2) where the membrane fusion peptide resides,and (iii) a transmembrane domain (HA2) that anchors hemagglutinin to theviral envelope. A set of amino acids in the interface of the HA1 and HA2peptides is highly conserved across all influenza subtypes. The HA1/HA2membrane proximal region (MPER), including a canonical alpha-helix, isalso highly conserved across influenza subtypes.

HA polypeptides interact with the surface of cells by binding to aglycoprotein receptor, known as the HA receptor. Binding of an HApolypeptide to an HA receptor is predominantly mediated by N-linkedglycans on the HA receptors. HA polypeptides on the surface of flu virusparticles recognize sialylated glycans that are associated with HAreceptors on the surface of the cellular host. Following replication ofviral proteins and genome by the cellular machinery, new viral particlesbud from the host to infect neighboring cells.

Currently, vaccines are administered to subjects, e.g., humans, toprevent the flu, e.g., to prevent infection or to minimize the effectsof an infection with influenza virus. Traditional vaccines contain acocktail of antigens from various strains of influenza and areadministered to humans to prevent the human from getting infected withthe virus. HA is the main target of influenza A-neutralizing antibodies,and HA undergoes continuous evolution driven by the selective pressureof the antibody response, which is primarily directed against themembrane-distal receptor-binding subdomain of the HA polypeptide. Thesubject, however, is protected only from strains that are identical to,or closely related to, the strains from which the antigens in thecocktail were derived. The human is still most vulnerable to infectionby other strains of the flu that were not included in the cocktail. Oneof the advantages of the antibodies provided herein is their ability tobind an epitope of HA that is conserved across multiple strains ofinfluenza A, and in embodiments influenza B. Thus, administration of ananti-HA antibody described herein will be more effective to protect anindividual from infection from a broader spectrum of influenza (e.g.,influenza A and, in embodiments, influenza B) and conditions associatethereof (e.g., secondary infections, e.g., secondary bacterialinfections). Further, the antibodies are effective in treating a subjectafter infection has occurred.

Anti-HA Antibody Molecules

Binding agents, and in particular, the antibody molecules describedherein, can bind to influenza A viruses from both Group 1 and Group 2,and in embodiments also bind influenza B viruses. For example, theantibody molecules described herein can bind to an HA polypeptide on atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 strains from Group 1, and canalso bind to an HA polypeptide on at least 1, 2, 3, 4, 5, or 6 strainsfrom Group 2. In another example, the antibody molecules describedherein can bind to an HA polypeptide on an influenza strain from atleast 1, 2 or 3 clades from Group 1, and can also bind to an HApolypeptide on an influenza strain from one or both clades of Group 2.The antibody molecules described herein inhibit cell entry and thustargeting an early step in the infection process.

The binding agents, and in particular, the antibody molecules featuredin the disclosure, can be effective to treat or prevent infection byseasonal or pandemic influenza strains. The binding agents, and inparticular the antibody molecules described herein, can be characterizedby their ability to prevent or treat a Group 1 or a Group 2 strain ofinfluenza A viruses or, in embodiments, a strain of influenza B viruses.The binding agents, and in particular the antibody molecules featured inthe disclosure, are effective to prevent or treat infection by one ormore strains of Group 1, one or more strains of Group 2, and also one ormore strains of influenza B viruses.

The binding agents, and in particular the antibody molecules can beeffective to treat the infection when administered the same day as thesubject is exposed, or when administered, e.g., 1 day, 2 days, 3 days, 4days or later after infection, or upon a first symptom experienced bythe patient.

Strains

The antibody molecules described herein are effective to treat one ormore influenza strains of Group 1, one or more influenza strains ofGroup 2, and also one or more influenza B strains, and specific isolateswithin these strains. Certain antibody molecules may be more effectivefor treatment of certain isolates than other isolates. Exemplaryinfluenza strains and isolates are described in the below Table 1.

TABLE 1 Exemplary influenza strains and Isolates Type Group HA typeIsolate A 1 H1N1 A/PR/8/34 (aka PR-8) A/Solomon Islands/03/06 A/SolomonIslands/20/1999 A/California/07/2009 A/New Caledonia/20/99A/Bangkok/10/83 A/Yamagata/120/86 A/Osaka/930/88 A/Suita/1/89A/California/04/2009 A 1 H2N2 A/Okuda/57 A/Adachi/2/57 A/Kumamoto/1/65A/Kaizuka/2/65 A/Izumi/5/65 A/Chicken/PA/2004 A 1 H5N1 A/Vietnam/1203/04A/Duck/Singapore/3/97 A/Duck/MN/1525/81 A 1 H9N2 A/Hong Kong/1073/2004A/Swine/Hong Kong/9/98 A/Guinea fowl/HK/WF10/99 A 1 H16N3 A/black headedgull/ Mongolia/1756/2006 A 2 H3N2 X-31 A/Victoria/3/75 A/Wyoming/03/2003A/Wisconsin/67/2005 A/Brisbane/10/2007 A/California/7/2004 A/NewYork/55/2004 A/Moscow/10/1999 A/Aichi/2/68 A/Beijing/32/92/X-117A/Fukuoka/C29/85 A/Sichuan/2/87 A/Ibaraki/1/90 A/Suita/1/90A/Perth/16/2009 A/Uruguay/716/2007 A/Fujian/411/2003 A/Panama/2007/99A/Shangdong/09/93 A 2 H7N7 A/Netherlands/219/2003 B B/Wisconsin/1/2010

Affinity can also be in reference to a particular isolate of a givenGroup 1 or Group 2 strain for influenza A viruses or a strain forinfluenza B viruses. Exemplary isolates are as provided in the aboveTable 1.

Mechanisms of Inhibition

While not being limited by a specific mechanism, HA specific antibodiescan inhibit infection by numerous methods, such as by blocking viralattachment to sialic acid residues on surface proteins on host cells, byinterfering with the structural transition of HA that triggers fusionactivity in the endosome, or by simultaneously inhibiting attachment andvirus-cell fusion.

In embodiments, antibody molecules featured herein bind an epitope atthe HA trimer interface. Structural changes at the trimer interface areimportant for fusion of the viral membrane and the endocytic membrane,and the antibody molecules described herein interfere with this criticalstep of infection. Assays to measure fusogenic activity of HA are knownin the art. For example, one fusion assay measures syncytia formation,which occurs in cell-cell fusion events. Cells that express and displayan influenza viral strain HA can be used in the assay. Membrane-anchoredhemagglutinin in these cells is induced to convert to the fusionconformation by a brief (e.g., 3 minute) exposure to low pH (e.g., pH5). A 2-3-hour incubation period follows to allow the cells to recoverand fuse to form syncytia. A nuclear stain can be used to aid in thevisualization of these fusion products, and their count is used as agauge of fusion activity. A candidate anti-HA antibody can be addedeither before or after the low pH treatment to determine at which stageof the fusion process the antibody interferes.

Another type of fusion assay monitors content mixing. To measure contentmixing, host cells (e.g., erythrocytes) are loaded with a dye (e.g.,Lucifer yellow) to determine whether the contents of HA-bound host cellscould be delivered to HA-expressing cells after exposure tofusion-inducing conditions (e.g., low pH, such as pH less than 6 or pHless than 5). If the dye fails to mix with the contents of the hostcells, then the conclusion can be made that fusion is inhibited. See,e.g., Kemble et al., J. Virol. 66:4940-4950, 1992.

In another example, a fusion assay is performed by monitoring lipidmixing. The lipid mixing assay can be performed by labeling host cells(e.g., erythrocytes) with a fluorescent dye (e.g., R18(octadecylrhodamine)) or dye pairs (e.g., CPT-PC/DABS-PC) (forfluorescence resonance energy transfer), exposing the host cells andHA-expressing cells to fusion-inducing conditions, and assaying forfluorescence dequenching (FDQ). Lipid mixing leads to dilution of thelabel into the viral envelope and a consequent dequenching. A lag indequenching or the absence of dequenching is indicative of membranefusion inhibition. See, e.g., Kemble et al., J. Virol. 66:4940-4950,1992; and Carr et al., Proc. Natl. Acad. Sci. 94:14306-14313, 1997.

Escape Mutants

In embodiments, influenza strains will rarely if ever produce escapemutants when contacted with the featured antibody molecules.

Escape mutants can be identified by methods known in the art. Forexample, an antibody featured in the disclosure will not produce anescape mutant when the cells are infected with the virus under prolongedor repeated exposure to anti-HA antibodies featured in the disclosure.

One exemplary method includes infection of cells (e.g. MDCK cells) witha fixed amount of influenza A viral particles in the presence of theantibody at a concentration known to attenuate infection rates by 50%.Viral progeny collected after each passaging is used to infect a freshcell culture in the presence of the same or greater concentration of theantibody. After multiple cycles of infection, e.g., after 15 cycles, 12cycles, 11 cycles, 10 cycles, 9 cycles, 8 cycles, 7 cycles, 6 cycles, or5 cycles, of infection under these conditions, the HA nucleotidesequence extracted from 20 viral plaque picks is evaluated forenrichment for mutations that renders the viral isolate resistant toneutralization by the antibody (an escape mutant). If no mutants withreduced sensitivity to the antibody are detected after the multiplerounds of selection, e.g., after 11 rounds, 10 rounds, or 9 rounds ofselection, the antibody is determined to be resistant to escapemutations (see, e.g., Throsby et al. (2008) PLoS One, volume 3, e3942).

In another example, an assay that measures minimum inhibitoryconcentration (MIC) of the neutralizing antibody can be used to identifyescape mutants. The MIC of an antibody molecule is the lowestconcentration of an antibody molecule that can be mixed with virus toprevent infection of cell culture with influenza. If escape mutantsarise within a viral population, then the MIC of a particular antibodywill be observed to increase with increased rounds of propagation underthe antibody selective pressure, as the proportion of the viralparticles that carry the resistance mutation within the populationincreased. Influenza escape mutants rarely if ever evolve in response toan anti-HA antibody molecule described herein, and therefore the MICwill stay the same over time.

Another assay suitable for monitoring for the development of escapemutants is a Cytopathic Effect (CPE) assay. A CPE assay monitors theability of an antibody to neutralize (i.e., prevent infection by) aninfluenza strain. A CPE assay provides the minimal concentration ofantibody required in cell culture to neutralize the virus. If escapemutants arise, than the CPE of a particular antibody will increase overtime, as the antibody becomes less effective at neutralizing the virus.Viral strains rarely if ever produce escape mutants in response to ananti-HA antibody molecule described herein, and therefore the CPE willstay essentially the same over time.

Quantitative polymerase chain reaction (qPCR) can also be used tomonitor for the development of escape mutants. qPCR is useful to monitorthe ability of an antibody to neutralize (i.e., prevent infection by) aninfluenza strain. If an antibody effectively neutralizes a virus, thenqPCR performed on cell culture samples will not detect presence of viralgenomic nucleic acid. If escape mutants arise, than over time, qPCR willamplify more and more viral genomic nucleic acid. Escape mutants rarelyif ever develop in response to an anti-HA antibody molecule describedherein, and therefore qPCR will rarely if ever detect viral genomicnucleic acid, even after the passage of time.

Binding and Affinity

In embodiments, the binding agents, particularly antibody molecules,featured herein bind to two or more of the following:

at least one HA polypeptide from a Group 1 influenza strain (e.g., anH1, H2, H5, H6, H8, H9 H12, H11, H13, H16 or H17 polypeptide);

at least one HA polypeptide from a Group 2 influenza strain (e.g., anH3, H4, H14, H7, H10, or H15 polypeptide); and

at least one HA polypeptide from a influenza B strain.

In an embodiment, a binding agent, e.g., an antibody molecule, will havea K_(D) for an HA from a Group 1 influenza strain (e.g., an H1, H2, H5,H6, H8, H9 H12, H11, H13, H16 or H17 polypeptide) of equal to or lessthan 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹².

In an embodiment, a binding agent, e.g., an antibody molecule, will havea K_(D) for an HA from a Group 2 influenza strain (e.g., an H3, H4, H14,H7, H10, or H15 polypeptide) of equal to or less than 10⁻⁶, 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹².

In an embodiment, a binding agent, e.g., an antibody molecule, will havea K_(D) for an influenza B HA of equal to or less than 10⁻⁶, 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹².

In an embodiment, a binding agent, e.g., an antibody molecule, willhave:

a) a first K_(D) (representing an affinity for an HA from a Group 1influenza strain, e.g., an H1, H2, H5, H6, H8, H9 H12, H11, H13, H16 orH17 polypeptide); and

b) a second K_(D) (representing an affinity for an HA from a Group 2influenza strain, e.g., an H3, H4, H14, H7, H10, or H15 polypeptide),

wherein the first and second K_(D) are one or both of:

both equal to or less than 10⁻⁸; and

within 10 or 100 fold of each other;

In an embodiment, a binding agent, e.g., an antibody molecule, will have

a) a first K_(D) (representing an affinity for an H1, e.g., the H1 froman an H1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934,or A/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004); and

b) a second K_(D) (representing an affinity for an H3 polypeptide, e.g.,the H3 from an H3N2 strain, e.g., A/Brisbane/59/2007),

wherein the first and second K_(D) are one or both of:

both equal to or less than 10⁻⁸; and

within 10 or 100 fold of each other;

In an embodiment, a binding agent, e.g., an antibody molecule, will have

a) a first K_(D) (representing an affinity for an H1, e.g., the H1 froman an H1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934,or A/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004); and

b) a second K_(D) (representing an affinity for an H3 polypeptide, e.g.,the H3 from an H3N2 strain, e.g., A/Brisbane/59/2007),

wherein the first and second K_(D) are one or both of:

both equal to or less than 10⁻⁸; and

within 10 or 100 fold of each other.

In an embodiment, a binding agent, e.g., an antibody molecule, willhave:

a) a first K_(D) (representing an affinity for an HA from a Group 1influenza strain, e.g., an H1, H2, H5, H6, H8, H9 H12, H11, H13, H16 orH17 polypeptide and/or an affinity for an HA from a Group 2 influenzastrain, e.g., an H3, H4, H14, H7, H10, or H15 polypeptide); and

b) a second K_(D) (representing an affinity for an influenza B HA, e.g.,from B/Wisconsin/1/2010);

wherein the first and second K_(D) are one or both of:

both equal to or less than 10⁻⁸; and

within 10 or 100 fold of each other.

In an embodiment, a binding agent, e.g., an antibody molecule, willhave:

a) a first K_(D) (representing an affinity for an HA from a Group 1influenza strain, e.g., an an H1, e.g., the H1 from an an H1N1 strain,e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004, and/or an affinity for an HA from a Group 2influenza strain, e.g., an H3 polypeptide, from an H3N2 strain, e.g.,from A/Brisbane/59/2007); and

b) a second K_(D) (an affinity for an influenza B HA);

wherein the first and second K_(D) are: one or both of:

both equal to or less than 10⁻⁸; and

within 10 or 100 fold of each other.

In one embodiment, the antibody molecule binds to at least one HApolypeptide from a Group 1 influenza strain with a higher affinity thana reference anti-HA antibody, and to at least one HA polypeptide from aGroup 2 influenza strain with a higher affinity than a reference anti-HAantibody. In another embodiment, the antibody molecule binds to at leastone HA polypeptide from an influenza A strain with a higher affinitythan a reference anti-HA antibody, and to at least one HA polypeptidefrom an influenza B strain with a higher affinity than a referenceanti-HA antibody. Exemplary reference HA antibodies include Ab 67-11(U.S. Provisional application No. 61/645,453, filed on the same date asthe present application), FI6 (FI6, as used herein, refers to anyspecifically disclosed FI6 sequence in U.S. Published Application No.2010/0080813, US published application No. 2011/0274702, WO2013/011347or Corti et al., Science 333:850-856, 2011, published online Jul. 28,2011; FIGS. 12A to 12C), FI28 (U.S. Published Application No.2010/0080813), and C179 (Okuno et al., J. Virol. 67:2552-1558, 1993),F10 (Sui et al., Nat. Struct. Mol. Biol. 16:265, 2009), CR9114 (Dreyfuset al., Science. 2012; 337(6100):1343-1348; published online Aug. 9,2012), and CR6261 (Ekiert et al., Science 324:246-251, 2009; publishedonline Feb. 26, 2009).

Affinity, or relative affinity or aviditiy, can be measured by methodsknown in the art, such as by ELISA assay (Enzyme Linked ImmunosorbentAssay), Surface Plasmon Resonance (SPR, e.g., by a Biacore™ Assay), orKinExA® assay (Sapidyne, Inc.). Relative binding affinity is expressedherein according to ELISA assay. As used herein, an anti-HA antibodythat binds with “high affinity” to a Group 1 HA, to a Group 2 HA, and toa influenza B HA, can bind a Group 1 HA with a Kd less than or equal to200 pM, e.g., less than or equal to 100 pM, as measured by ELISA, canbind a Group 2 HA with a Kd less than or equal to 200 pM, e.g., lessthan or equal to 100 pM, as measured by ELISA, and can bind an influenzaB HA with a Kd less than or equal to 200 pM, e.g., less than or equal to100 pM, as measured by ELISA.

Exemplary Anti-HA Antibody Molecules

Provided herein are antibodies that have one or more CDR sequences andone or more framework (FR) sequences as shown in Table 2.

TABLE 2 Heavy and Light Chain CDR and FR Sequences for Anti-HAAntibodies. CDR/FR SEQ ID Region Amino Acid Sequence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

In one embodiment, the anti-HA antibody comprises a heavy chain and/or alight chain as defined in Table 3 below. The amino acid sequences of thevariable heavy and light chains of Table 3 are provided in FIGS. 2, 3,respectively, or in FIG. 17.

TABLE 3 Heavy and Light Chain Amino Acid Sequence Designations forAnti-HA Antibodies Antibody HC SEQ ID NO: LC SEQ ID NO: 1. Ab A18 15 1528 28 2. Ab 014 16 16 29 29 3. Ab 028 16 16 30 30 4. Ab 001 17 17 31 315. Ab 002 18 18 31 31 6. Ab 003 19 19 31 31 7. Ab 009 17 17 32 32 8. Ab010 18 18 32 32 9. Ab 011 19 19 32 32 10. Ab 017 17 17 33 33 11. Ab B1818 18 33 33 12. Ab 019 19 19 33 33 13. Ab 025 17 17 34 34 14. Ab 026 1818 34 34 15. Ab 027 19 19 34 34 16. Ab 086 20 20 34 34 17. Ab 154 21 2129 29 18. Ab 155 21 21 30 30 19. Ab 157 22 22 29 29 20. Ab 159 22 22 3535 21. Ab 160 17 17 36 36 22. Ab 186 17 17 37 37 23. Ab 187 17 17 38 3824. Ab 188 17 17 39 39 25. Ab 189 17 17 40 40 26. Ab 190 17 17 41 41 27.Ab 191 17 17 42 42 28. Ab 192 17 17 43 43 29. Ab 193 17 17 44 44 30. Ab194 19 19 37 37 31. Ab 195 19 19 38 38 32. Ab 196 19 19 39 39 33. Ab 19719 19 40 40 34. Ab 198 19 19 41 41 35. Ab 199 19 19 42 42 36. Ab 200 1919 43 43 37. Ab 202 17 17 45 45 38. Ab 203 18 18 45 45 39. Ab 204 19 1945 45 40. Ab 210 23 23 45 45 41. Ab 211 17 17 46 46 42. Ab 212 18 18 4646 43. Ab 213 19 19 46 46 44. Ab 219 23 23 46 46 45. Ab A001 24 24 47 4746. Ab A002 24 24 48 48 47. Ab A003 24 24 49 49 48. Ab 004 25 25 47 4749. Ab 005 25 25 48 48 50. Ab 006 25 25 49 49 51. Ab 007 26 26 47 47 52.Ab 008 26 26 48 48 53. Ab A009 26 26 49 49 54. Ab A010 24 24 50 50 55.Ab A011 24 24 51 51 56. Ab 012 25 25 50 50 57. Ab 013 25 25 51 51 58. AbA14 26 26 50 50 59. Ab 015 26 26 51 51 60. Ab 016 27 27 47 47 61. AbA017 27 27 48 48 62. Ab C18 27 27 49 49 63. Ab A019 27 27 50 50 64. Ab031 24 24 45 45 65. Ab 032 25 25 45 45 66. Ab 033 26 26 45 45 67. Ab 03427 27 45 45 68. Ab 037 24 24 46 46 69. Ab 038 25 25 46 46 70. Ab 039 2626 46 46 71. Ab 040 27 27 46 46 72. Ab 043 25 25 60 60 73. Ab 044 25 2552 52 74. Ab 045 25 25 57 57 75. Ab 046 25 25 59 59 76. Ab 047 25 25 5555 77. Ab 048 25 25 58 58 78. Ab 049 25 25 54 54 79. Ab 050 25 25 56 5680. Ab 051 25 25 53 53 81. Ab 052 25 25 61 61 82. Ab 067 25 25 153 15383. Ab 068 25 25 154 154 84. Ab 069 25 25 155 155 85. Ab 070 25 25 156156 86. Ab 071 162 162 52 52 87. Ab 072 163 163 52 52 88. Ab 073 25 25165 165 89. Ab 074 25 25 166 166 90. Ab 075 25 25 167 167 91. Ab 076 2525 168 168 92. Ab 077 25 25 169 169 93. Ab 078 164 164 52 52 94. Ab 079164 164 155 155 95. Ab 080 164 164 166 166 96. Ab 081 164 164 169 169

In one embodiment, the anti-HA antibody comprises a heavy chain asdefined in Table 4A below, and/or a light chain as defined in Table 4Abelow.

TABLE 4A Heavy and Light Chain Amino Acid Sequence Designations HC SEQID NO: LC SEQ ID NO: 15 15 28 28 16 16 29 29 17 17 30 30 18 18 35 35 1919 31 31 21 21 32 32 22 22 33 33 20 20 34 34 23 23 36 36 24 24 45 45 2525 46 46 26 26 37 37 27 27 38 38 Hc consensus 161 39 39 (HC161) 162 16240 40 163 163 41 41 164 164 42 42 43 43 44 44 47 47 48 48 49 49 50 50 5151 52 52 53 53 54 54 55 55 56 56 57 57 58 58 59 59 60 60 61 61 153 153154 154 155 155 156 156 LC consensus 62 (LC62) 165 165 166 166 167 167168 168 169 169

In one embodiment, an antibody featured in the disclosure comprises aheavy chain sequence as defined in Table 4A and a light chain sequenceas defined in Table 4A.

In one embodiment, an antibody featured in the disclosure comprises aheavy chain sequence as defined herein, e.g., in Table 4A, where adipeptide is fused to the N-terminus. Typically, the dipeptide isisoleucine-aspartic acid (Ile-Asp). In another embodiment, an antibodyfeatured in the disclosure comprises a light chain sequence as definedherein, e.g., in Table 4A, where a dipeptide is fused to the N-terminus.Typically, the dipeptide is Ile-Asp. In yet another embodiment, anantibody featured in the disclosure comprises a heavy chain comprisingan N-terminal Ile-Asp dipeptide and a light chain comprising an Ile-Aspdipeptide. In the propeptide sequence of the heavy chain or light chainpolypeptide, the Ile-Asp dipeptide occurs between the signal sequenceand FR1. Heavy chain and light chain variable sequences comprising anIle-Asp dipeptide at the N-terminus are identified in Table 4B.

TABLE 4B Heavy and Light Chain Amino Acid Sequence Designations, wherethe Sequence Includes an N-terminal Ile-Asp Dipeptide HC SEQ ID NO: LCSEQ ID NO: 15-ID 96 28-ID 110 16-ID 97 29-ID 111 17-ID 98 30-ID 11218-ID 99 35-ID 113 19-ID 100 31-ID 114 21-ID 101 32-ID 115 22-ID 10233-ID 116 20-ID 103 34-ID 117 23-ID 104 36-ID 118 24-ID 105 45-ID 11925-ID 106 46-ID 120 26-ID 107 37-ID 121 27-ID 108 38-ID 122 Hc consensus109 39-ID 123 ID (161-ID) 40-ID 124 41-ID 125 42-ID 126 43-ID 127 44-ID128 47-ID 129 48-ID 130 49-ID 131 50-ID 132 51-ID 133 52-ID 134 53-ID135 54-ID 136 55-ID 137 56-ID 138 57-ID 139 58-ID 140 59-ID 141 60-ID142 61ID 143 153-ID  157 154-ID  158 155-ID  159 156-ID  160 LCconsensus 144 ID (62-ID)

In another embodiment, an antibody featured in the disclosure is otherthan an antibody known in the art. For example, the antibody is not Ab67-11 (U.S. Provisional application No. 61/645,453) FI6 (FI6, as usedherein, refers to any specifically disclosed FI6 sequence in U.S.Published Application No. 2010/0080813, US published application No.2011/0274702, WO2013/011347 or Corti et al., Science 333:850-856, 2011,published online Jul. 28, 2011; FIGS. 12A to 12C) FI28 (U.S. PublishedApplication No. 2010/0080813), C179 (Okuno et al., J. Virol. 67:2552,1993), F10 (Sui et al., Nat. Struct. Mol. Biol. 16:265, 2009), CR9114(Dreyfus et al., Science 337:1343, 2012), or CR6261 (Ekiert et al.,Science 324:246, 2009).

In one embodiment, an antibody featured in the disclosure is other thanAb 67-11 (U.S. Provisional application No. 61/645,453, filed on the samedate as the present application).

Variants

In an embodiment, an antibody molecule, e.g., an antibody featured inthe disclosure has a variable heavy chain immunoglobulin domain that isat least 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%homologous, or at least 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%,97%, 98%, or 99% identical, to a heavy chain disclosed herein, e.g.,from Table 3, Table 4A, Table 4B, FIG. 2, FIG. 13 or FIG. 17, e.g.consensus sequence of SEQ ID NO:161, and has a variable light chainimmunoglobulin domain that is at least 85%, 87%, 88%, 89%, 90%, 92%,94%, 95%, 96%, 97%, 98%, or 99% homologous, or at least 85%, 87%, 88%,89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to a lightchain disclosed herein, e.g., from Table 3, Table 4A, Table 4B, FIG. 3,FIG. 14 or FIG. 17, e.g., the consensus sequence of SEQ ID NO:62. Theconsensus sequences were determined through the analysis of biochemicaland biophysical properties of several hundred computationally designedVH/VL combinations. The consensus sequences represent the amino acidsequences in which each amino acid is the one that occurs mostfrequently at that site when multiple sequences comprising desirablebiochemical and biophysical data are aligned.

An exemplary anti-HA binding antibody has one or more CDRs, e.g., allthree HC CDRs and/or all three LC CDRs of a particular antibodydisclosed herein, or CDRs that are, in sum, at least 85%, 87%, 88%, 89%,90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homologous, or at least 85%,87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical, tosuch an antibody.

In one embodiment, the H1 and H2 hypervariable loops have the samecanonical structure as those of an antibody described herein. In oneembodiment, the L1 and L2 hypervariable loops have the same canonicalstructure as those of an antibody described herein.

In one embodiment, the amino acid sequence of the HC and/or LC variabledomain sequence is at least 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%,97%, 98%, or 99% homologous, or at least 85%, 87%, 88%, 89%, 90%, 92%,94%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence ofthe HC and/or LC variable domain of an antibody described herein. Theamino acid sequence of the HC and/or LC variable domain sequence candiffer by at least one amino acid, but no more than ten, eight, six,five, four, three, or two amino acids from the corresponding sequence ofan antibody described herein. For example, the differences may beprimarily or entirely in the framework regions.

In certain embodiments, the amino acid differences are conservativeamino acid differences (e.g., conservative amino acid substitutions). A“conservative” amino acid substitution is one in which the amino acidresidue is replaced with an amino acid residue comprising a similar sidechain. Families of amino acid residues comprising similar side chainshave been defined in the art. These families include, e.g., amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

The amino acid sequences of the HC and LC variable domain sequences canbe encoded by a nucleic acid sequence that hybridizes under highstringency conditions to a nucleic acid sequence described herein or onethat encodes a variable domain or an amino acid sequence describedherein. In one embodiment, the amino acid sequences of one or moreframework regions (e.g., FR1, FR2, FR3, and/or FR4) of the HC and/or LCvariable domain are at least 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%,96%, 97%, 98%, or 99% homologous, or at least 85%, 87%, 88%, 89%, 90%,92%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to correspondingframework regions of the HC and LC variable domains of an antibodydescribed herein. In one embodiment, one or more heavy or light chainframework regions (e.g., HC FR1, FR2, and FR3) are at least 85%, 87%,88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homologous, or atleast 85%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%identical, to the sequence of corresponding framework regions from ahuman germline antibody.

Validation of Epitopes

In one embodiment, the antibodies featured in the disclosure are usefulfor validating a vaccine based on a particular epitope. For example, anepitope that is the target of an antibody featured in the disclosure canbe assessed by computation methods to identify a peptide frameworksuitable for supporting the epitope conformation, such as to stabilizean epitope that is transient or minimally accessible in nature.Computational abstraction of the epitope and framework properties allowsautomated screening of databases to identify candidate acceptor peptidescaffolds. The acceptor scaffold can have a particular tertiarystructure that includes, for example, one or more of a beta sheet, abeta sandwich, a loop, or an alpha or beta helix. The candidateepitope-scaffold antigens can be assayed in vitro, such as to identifybinding properties with an antibody featured in the disclosure, e.g.,binding affinity or structure analysis of the epitope-scaffold/antibodycomplex, or in vitro neutralization. The ability of the epitope-scaffoldto generate an immune response (e.g., to generate antibodies) can betested by administering the epitope-scaffold to an animal (e.g., in amammal, such as a rat, a mouse, a guinea pig, or a rabbit), and thentesting sera for the presence of anti-epitope-scaffold antibodies, e.g.,by ELISA assay. The ability of the epitope-scaffold to elicit protectionagainst infection by an influenza A Group 1 or Group 2 strain, or byboth types of influenza strains, or an influenza B strain, can beassessed in vivo, such as in an animal (e.g., in a mammal). Thus, anantibody featured in the disclosure can provide validation that theepitope is functionally important and that targeting the epitope willprovide protection from infection with a Group 1 or Group 2 influenzastrain, or both types of strains, or an influenza B strain.

Production of Antibody Molecules

The nucleic acids (e.g., the genes) encoding an antibody moleculegenerated by a method described herein can be sequenced, and all or partof the nucleic acids can be cloned into a vector that expresses all orpart of the nucleic acids. For example, the nucleic acids can include afragment of the gene encoding the antibody, such as a single chainantibody (scFv), a F(ab′)₂ fragment, a Fab fragment, or an Fd fragment.

The disclosure also provides host cells comprising the nucleic acidsencoding an antibody or fragment thereof as described herein. The hostcells can be, for example, prokaryotic or eukaryotic cells, e.g.,mammalian cells, or yeast cells, e.g., Pichia (see, e.g., Powers et al.(2001) J. Immunol. Methods 251:123-35), Hanseula, or Saccharomyces.

Antibody molecules, particularly full length antibody molecules, e.g.,IgGs, can be produced in mammalian cells. Exemplary mammalian host cellsfor recombinant expression include Chinese Hamster Ovary (CHO) cells(including dhfr CHO cells, described in Urlaub and Chasin (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COScells, K562, and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulindomain, the recombinant expression vectors may carry additional nucleicacid sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use in dhfr⁻host cells with methotrexate selection/amplification) and the neo gene(for G418 selection).

In an exemplary system for recombinant expression of an antibodymolecule (e.g., a full length antibody or an antigen-binding portionthereof), a recombinant expression vector encoding both the antibodyheavy chain and the antibody light chain is introduced into dhfr− CHOcells by calcium phosphate-mediated transfection. Within the recombinantexpression vector, the antibody heavy and light chain genes are eachoperatively linked to enhancer/promoter regulatory elements (e.g.,derived from SV40, CMV, adenovirus and the like, such as a CMVenhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLPpromoter regulatory element) to drive high levels of transcription ofthe genes. The recombinant expression vector also carries a DHFR gene,which allows for selection of CHO cells that have been transfected withthe vector using methotrexate selection/amplification. The selectedtransformant host cells are cultured to allow for expression of theantibody heavy and light chains and intact antibody molecule isrecovered from the culture medium. Standard molecular biology techniquesare used to prepare the recombinant expression vector, to transfect thehost cells, to select for transformants, to culture the host cells, andto recover the antibody from the culture medium. For example, someantibodies can be isolated by affinity chromatography with a Protein Aor Protein G. For example, purified antibodies can be concentrated toabout 100 mg/mL to about 200 mg/mL using protein concentrationtechniques that are known in the art.

Antibody molecules can also be produced by a transgenic animal. Forexample, U.S. Pat. No. 5,849,992 describes a method for expressing anantibody molecule in the mammary gland of a transgenic mammal. Atransgene is constructed that includes a milk-specific promoter andnucleic acid sequences encoding the antibody molecule of interest, e.g.,an antibody described herein, and a signal sequence for secretion. Themilk produced by females of such transgenic mammals includes, secretedtherein, the antibody of interest, e.g., an antibody described herein.The antibody molecule can be purified from the milk, or for someapplications, used directly.

Antibody molecules can also be expressed in vivo, followingadministration of a vector containing nucleic acids encoding theantibody heavy chain and the antibody light chain. Vector mediatedgene-transfer is then used to engineer secretion of the anti-HA antibodyinto circulation. For example, an anti-HA antibody heavy chain and ananti-HA antibody light chain as described herein are cloned into anadeno-associated virus (AAV)-based vector, and each of the anti-HAantibody heavy chain and the anti-HA antibody light chain are undercontrol of a promoter, such as a cytomegalovirus (CMV) promoter.Administration of the vector to a subject, such as to a patient, e.g., ahuman patient, such as by intramuscular injection, results in expressionof an anti-HA antibody, and secretion into the circulation.

Modifications of Binding Agents

Binding, agents, e.g., antibody molecules can be modified to havenumerous properties, e.g., to have altered, e.g., extended half life, tobe associated with, e.g., covalently bound to detectable moieties, e.g.,labels, to be associated with, e.g., covalently bound to toxins, or tohave other properties, e.g., altered immune fucntions.

Antibody molecules may include modifications, e.g., modifications thatalter Fc function, e.g., to decrease or remove interaction with an Fcreceptor or with C1q, or both. In one example, the human IgG1 constantregion can be mutated at one or more residues.

For some antibody molecules that include an Fc domain, the antibodyproduction system may be designed to synthesize antibody molecules inwhich the Fc region is glycosylated. The Fc domain can be produced in amammalian expression system that appropriately glycosylates the residuecorresponding to asparagine 297. The Fc domain can also include othereukaryotic post-translational modifications.

Other suitable Fc domain modifications include those described inWO2004/029207. For example, the Fc domain can be an XmAb® Fc (Xencor,Monrovia, Calif.). The Fc domain, or a fragment thereof, can have asubstitution in an Fcγ Receptor (FcγR) binding region, such as thedomains and fragments described in WO05/063815. In some embodiments, theFc domain, or a fragment thereof, has a substitution in a neonatal FcReceptor (FcRn) binding region, such as the domains and fragmentsdescribed in WO05047327. In other embodiments, the Fc domain is a singlechain, or fragment thereof, or modified version thereof, such as thosedescribed in WO2008143954. Other suitable Fc modifications are known anddescribed in the art.

Antibody molecules can be modified, e.g., with a moiety that improvesits stabilization and/or retention in circulation, e.g., in blood,serum, lymph, bronchoalveolar lavage, or other tissues, e.g., by atleast 1.5, 2, 5, 10, or 50 fold.

For example, an antibody molecule generated by a method described hereincan be associated with a polymer, e.g., a substantially non-antigenicpolymer, such as a polyalkylene oxide or a polyethylene oxide. Suitablepolymers will vary substantially by weight. Polymers comprisingmolecular number average weights ranging from about 200 to about 35,000daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) canbe used.

For example, an antibody molecule generated by a method described hereincan be conjugated to a water soluble polymer, e.g., a hydrophilicpolyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone. Anon-limiting list of such polymers include polyalkylene oxidehomopolymers such as polyethylene glycol (PEG) or polypropylene glycols,polyoxyethylenated polyols, copolymers thereof and block copolymersthereof, provided that the water solubility of the block copolymers ismaintained. Additional useful polymers include polyoxyalkylenes such aspolyoxyethylene, polyoxypropylene, and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides that comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparan.

Binding agents, e.g., antibody molecules, as disclosed herein, can byconjugated to another entity or moiety (e.g., to a cytotoxic orcytostatic moiety, a label or detectable moiety, or a therapeuticmoiety). Exemplary moieties include: a cytotoxic or cytostatic agent,e.g., a therapeutic agent, a drug, a compound emitting radiation,molecules of plant, fungal, or bacterial origin, or a biological protein(e.g., a protein toxin) or particle (e.g., a recombinant viral particle,e.g., via a viral coat protein), a detectable agent; a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag). A binding agent, e.g.,an antibody molecule, as disclosed herein, can be functionally linked byany suitable method (e.g., chemical coupling, genetic fusion, covalentbinding, noncovalent association or otherwise) to one or more othermolecular entities.

Binding agents, e.g., antibody molecules, disclosed herein can beconjugated with a detectable moiety, e.g., a label or imaging agent.Such moieties can include enzymes (e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase,acetylcholinesterase, glucose oxidase and the like), radiolabels (e.g.,³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I and the like), haptens,fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors,fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike), phosphorescent molecules, chemiluminescent molecules,chromophores, luminescent molecules, photoaffinity molecules, coloredparticles or affinity ligands, such as biotin, predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, or binding sites for secondary antibodies, metal bindingdomains, epitope tags). In some embodiments, a moiety, e.g., adetectable moiety, e.g., a label, is attached by spacer arms of variouslengths to reduce potential steric hindrance.

In embodiments a binding agent, e.g., antibody molecule, disclosedherein, is derivatized with a detectable enzyme and is detected byadding additional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. A binding agent, e.g., antibody molecule, disclosed herein,ay also be derivatized with a prosthetic group (e.g.,streptavidin/biotin and avidin/biotin). For example, an antibody may bederivatized with biotin, and detected through indirect measurement ofavidin or streptavidin binding.

In embodiments the moiety comprises paramagnetic ions and NMR-detectablesubstances, among others. For example, in some embodiments, aparamagnetic ion is one or more of chromium (III), manganese (II), iron(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III), erbium (III), lanthanum(III), gold (III), lead (II), and/or bismuth (III).

Binding agents, e.g., antibody molecules, as disclosed herein, can bemodified to be associated with, e.g., conjugated to, a therapeuticagent, e.g., an agent comprising anti-viral activity, anti-inflammatoryactivity, or cytotoxic activity, etc. In some embodiments, therapeuticagents can treat symptoms or causes of influenza infection (e.g., forexample, anti-viral, pain-relief, antiinflammatory, immunomodulatory,sleep-inducing activities, etc).

Treatment Methods and Administration

The binding agents, e.g., antibody molecules, featured in thedisclosure, can be used to treat a subject, e.g., a subject, e.g., ahuman subject, infected with, or at risk for becoming infected with, aninfluenza virus.

Any human is candidate to receive an antibody molecule featured in thedisclosure for treatment or prevention of an infection by an influenzavirus. Humans at high risk of infection, such as immunocompromisedindividuals, and humans who are at high risk of exposure to influenzavirus are particularly suited to receive treatment with the antibodymolecule Immunocompromised individuals include the elderly (65 years andolder) and children (e.g., 6 months to 18 years old), and people withchronic medical conditions. People at high risk of exposure includeheath care workers, teachers and emergency responders (e.g.,firefighters, policemen).

The antibody molecules described herein can also be used to prevent orreduce (e.g., minimize) secondary infection (e.g., secondary bacterialinfection) or a risk of comprising secondary infection associated withinfluenza, or any effects (e.g., symptoms or complications) thereof on asubject. Opportunistic secondary bacterial infections (e.g., secondarybacterial pneumonia, e.g., primarily with Streptococcus pneumonia)contribute significantly to the overall morbidity and mortalityassociated with seasonal and pandemic influenza infections. The antibodymolecules described herein can be used to prevent or reduce (e.g.,minimize) the complications from secondary, opportunistic infections(e.g., bacterial infections) in a subject.

An antibody molecule can be administered to a subject, e.g., a humansubject, by a variety of methods. For many applications, the route ofadministration is one of: intravenous injection or infusion,subcutaneous injection, or intramuscular injection. An antibody moleculecan be administered as a fixed dose, or in a mg/kg dose. The antibodymolecule can be administered intravenously (IV) or subcutaneously (SC).For example, the antibody molecule can be administered at a fixed unitdose of between about 50-600 mg IV, e.g., every 4 weeks, or betweenabout 50-100 mg SC (e.g., 75 mg), e.g., at least once a week (e.g.,twice a week). In one embodiment, the antibody molecule is administeredIV at a fixed unit dose of 50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 130 mg,140 mg, 150 mg, 160 mg, 180 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600mg or more. Administration of the IV dose can be once or twice or threetimes or more per week, or once every two, three, four, or five weeks,or less frequently.

In one embodiment, the antibody molecule is administered SC at a fixedunit dose of 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or 120 mg ormore. Administration of the SC dose can be once or twice or three timesor more per week, or once every two, three, four, or five weeks, or lessfrequently.

An anti-HA antibody featured in the disclosure can also be administeredby inhalation, such as by intranasal or by oral inhalation, such as at afixed unit dose of 50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 130 mg, 140 mg,150 mg, 160 mg, 180 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg ormore.

In one embodiment, an anti-HA antibody is administered to a subject viavector-mediated gene transfer, such as through the delivery of a vectorencoding the heavy chain and the light chain of an anti-HA antibody, andthe antibody is expressed from the heavy chain and light chain genes inthe body. For example, nucleic acids encoding a heavy chain and a lightchain can be cloned in a AAV vector, such as a self-complementary AAVvector, the scAAV vector administered to a human by injection, such asby IM injection, and the antibody is expressed and secreted into thecirculation of the human

An antibody molecule can also be administered in a bolus at a dose ofbetween about 1 and 50 mg/kg, e.g., between about 1 and 10 mg/kg,between about 1 and 25 mg/kg or about 25 and 50 mg/kg, e.g., about 50mg/kg, 25 mg/kg, 10 mg/kg, 6.0 mg/kg, 5.0 mg/kg, 4.0 mg/kg, 3.0 mg/kg,2.0 mg/kg, 1.0 mg/kg, or less. Modified dose ranges include a dose thatis less than about 3000 mg/subject, about 1500 mg/subject, about 1000mg/subject, about 600 mg/subject, about 500 mg/subject, about 400mg/subject, about 300 mg/subject, about 250 mg/subject, about 200mg/subject, or about 150 mg/subject, typically for administration everyfourth week or once a month. The antibody molecule can be administered,for example, every three to five weeks, e.g., every fourth week, ormonthly.

Dosing can be adjusted according to a patient's rate of clearance of aprior administration of the antibody. For example, a patient may not beadministered a second or follow-on dose before the level of antibodiesin the patient's system has dropped below a pre-determined level. In oneembodiment, a sample from a patient (e.g., plasma, serum, blood, urine,or cerebrospinal fluid (CSF)) is assayed for the presence of antibodies,and if the level of antibodies is above a pre-determined level, thepatient will not be administered a second or follow-on dose. If thelevel of antibodies in the patient's system is below a pre-determinedlevel, then the patient is administered a second or follow-on dose. Apatient whose antibody levels are determined to be too high (above thepre-determined level) can be tested again after one or two or threedays, or a week, and if the level of antibody in the patient samples hasdropped below the pre-determined level, the patient may be administereda second or follow-on dose of antibody.

In certain embodiments, the antibody may be prepared with a carrier thatwill protect the drug against rapid release, such as a controlledrelease formulation, including implants, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known. See, e.g.,Controlled Drug Delivery (Drugs and the Pharmaceutical Sciences), SecondEdition, J. Robinson and V. H. L. Lee, eds., Marcel Dekker, Inc., NewYork, 1987.

Pharmaceutical compositions can be administered with a medical device.For example, pharmaceutical compositions can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modules arediscussed in, e.g., U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system comprising multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Of course,many other such implants, delivery systems, and modules are also known.

In embodiments the binding agent, e.g., an antibody molecule, isadministered buccally, orally, or by nasal delivery, e.g., as a liquid,spray, or aerosol, e.g., by topical application, e.g., by a liquid ordrops, or by inhalation.

An antibody molecule described herein can be administered with one ormore additional therapeutic agents, e.g., a second drug, for treatmentof a viral infection, or a symptom of the infection. The antibodymolecule and the one or more second or additional agents can beformulated together, in the same formulation, or they can be in separateformulations, and administered to a patient simultaneously orsequentially, in either order.

Dosage regimens are adjusted to provide the desired response, such as atherapeutic response or a combinatorial therapeutic effect. Generally,any combination of doses (either separate or co-formulated) of anantibody molecule and a second or additional agent can be used in orderto provide a subject with both agents in bioavailable quantities.

Dosage unit form or “fixed dose” as used herein refers to physicallydiscrete units suited as unitary dosages for the subjects to be treated;each unit contains a predetermined quantity of active compoundcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier and optionally in association withanother agent.

A pharmaceutical composition may include a “therapeutically effectiveamount” of an agent described herein. In embodiments where the antibodymolecule is administered in combination with a second or additionalagent, such effective amounts can be determined based on thecombinatorial effect of the administered first and second or additionalagent. A therapeutically effective amount of an agent may also varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the compound to elicit a desiredresponse in the individual, such as amelioration of at least oneinfection parameter, or amelioration of at least one symptom of theinfection, such as chills, fever, sore throat, muscle pain, headache,coughing, weakness, fatigue and general discomfort. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the composition are outweighed by the therapeutically beneficialeffects.

In an embodiment, administration of a binding agent, e.g., antibodymolecule, provided, e.g., as a pharmaceutical preparation, is by one ofthe following routes: oral, intravenous, intramuscular, intra-arterial,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by liquids, powders,ointments, creams, sprays, or drops), mucosal, nasal, buccal, enteral,sublingual; intratracheal instillation, bronchial instillation, and/orinhalation; and/or as an oral spray, nasal spray, and/or aerosol.

Combination Treatments and Exemplary Second or Additional Agents

Binding agents, e.g., antibody molecules, provided e.g., aspharmaceutical compositions, can be administered either alone or incombination with one or more other therapy, e.g., the administration ofa second or additional therapeutic agent.

In embodiments the combination can result in a lower dose of theantibody molecule or of the other therapy being needed, which, inembodiments can reduce side effects. In embodiments the combination canresult in enhanced delivery or efficacy of one or both agents. Theagents or therapies can be administered at the same time (e.g., as asingle formulation that is administered to a patient or as two separateformulations administered concurrently) or sequentially in any order.

Such second or additional agents include vaccines, anti-viral agents,and/or additional antibodies. In typical embodiments the second oradditional agent is not co-formulated with the binding agent, e.g.,antibody molecule, though in others it is.

In embodiments the binding agent, e.g., antibody molecule, and thesecond or additional agent are administered such that one or more of thefollowing is achieved: therapeutic levels, or therapeutic effects, ofone overlap the other; detectable levels of both are present at the sametime; or the therapeutic effect is greater than what would be seen inthe absence of either the binding agent, e.g., antibody molecule, or thesecond or additional agent. In embodiments each agent will beadministered at a dose and on a time schedule determined for that agent.

The second or additional agent can be, for example, for treatment orprevention of influenza. For example, the binding agents, e.g., antibodymolecules, e.g., therapeutic antibodies, provided herein can beadministered in combination with a vaccine, e.g., a vaccine describedherein or a mixture (a.k.a. a cocktail) of influenza peptides tostimulate the patient's immune system to prevent infection withparticular strains of influenza A. In other examples, the second oradditional agent is an anti-viral agent (e.g., an anti-NA or anti-M2agent), a pain reliever, an anti-inflammatory, an antibiotic, asteroidal agent, a second therapeutic antibody molecule (e.g., ananti-HA antibody), an adjuvant, a protease or glycosidase (e.g.,sialidase), etc.

Exemplary anti-viral agents include, e.g., vaccines, neuraminidaseinhibitors or nucleoside analogs. Exemplary anti-viral agents caninclude, e.g., zidovudine, gangcyclovir, vidarabine, idoxuridine,trifluridine, foscarnet, acyclovir, ribavirin, amantadine, remantidine,saquinavir, indinavir, ritonavir, alpha-interferons and otherinterferons, a neuraminidase inhibitor (e.g., zanamivir (Relenza®),oseltamivir (Tamiflu®), laninamivir, peramivir), rimantadine. Exemplarysecond antibody molecules include, for exampleAb 67-11 (U.S. Provisionalapplication No. 61/645,453, FI6 (U.S. Published Application No.2010/0080813), FI28 (U.S. Published Application No. 2010/0080813), C179(Okuno et al., J. Virol. 67:2552-8, 1993), F10 (Sui et al., Nat. Struct.Mol. Biol. 16:265, 2009), CR9114 (Dreyfus et al., Science 337:1343,2012), or CR6261 (see, e.g., Ekiert et al., Science 324:246, 2009).Thus, Ab 044 can be used in combination of any of those antibodies. Inother embodiments, two or more binding agents, e.g., antibody moleculesdisclosed herein, can be administered in combination, e.g., Ab 044 canbe administered in combination with Ab 032. In the case of combinations,two agents can be administered as part of the same dosage unit oradministered separately. Other exemplary agents useful for treating thesymptoms associated with influenza infection are acetaminophen,ibuprofen, aspirin, and naproxen.

In one embodiment, the antibody molecule and the second or additionalagent are provided as a co-formulation, and the co-formulation isadministered to the subject. It is further possible, e.g., at least 24hours before or after administering the co-formulation, to administerseparately one dose of the antibody formulation and then one dose of aformulation containing a second or additional agent. In anotherimplementation, the antibody molecule and the second or additional agentare provided as separate formulations, and the step of administeringincludes sequentially administering the antibody molecule and the secondor additional agent. The sequential administrations can be provided onthe same day (e.g., within one hour of one another or at least 3, 6, or12 hours apart) or on different days.

In embodiments the antibody molecule and the second or additional agentare each administered as a plurality of doses separated in time. Theantibody molecule and the second or additional agent are generally eachadministered according to a regimen. The regimen for one or both mayhave a regular periodicity. The regimen for the antibody molecule canhave a different periodicity from the regimen for the second oradditional agent, e.g., one can be administered more frequently than theother. In one implementation, one of the antibody molecule and thesecond or additional agent is administered once weekly and the otheronce monthly. In another implementation, one of the antibody moleculeand the second or additional agent is administered continuously, e.g.,over a period of more than 30 minutes but less than 1, 2, 4, or 12hours, and the other is administered as a bolus. In embodimentssequential administrations are administered. The time betweenadministration of the one agent and another agent can be minutes, hours,days, or weeks. The use of an antibody molecule described herein canalso be used to reduce the dosage of another therapy, e.g., to reducethe side-effects associated with another agent that is beingadministered. Accordingly, a combination can include administering asecond or additional agent at a dosage at least 10, 20, 30, or 50% lowerthan would be used in the absence of the antibody molecule. The antibodymolecule and the second or additional agent can be administered by anyappropriate method, e.g., subcutaneously, intramuscularly, orintravenously.

In some embodiments, each of the antibody molecule and the second oradditional agent is administered at the same dose as each is prescribedfor monotherapy. In other embodiments, the antibody molecule isadministered at a dosage that is equal to or less than an amountrequired for efficacy if administered alone. Likewise, the second oradditional agent can be administered at a dosage that is equal to orless than an amount required for efficacy if administered alone.

In some cases, the formulations described herein, e.g., formulationscontaining an antibody molecule featured in the disclosure, include oneor more second or additional agents, or are administered in combinationwith a formulation containing one or more second or additional agents.

In an embodiment a binding agent, e.g., antibody molecule, provided,e.g., as a pharmaceutical preparation, is administered by inhalation oraerosol delivery of a plurality of particles, e.g., particles comprisinga mean particle size of 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 microns.

Pharmaceutical Compositions

The binding agents, e.g., antibody molecules, featured in the disclosurecan be formulated as pharmaceutical compositions, such as for thetreatment or prevention of influenza.

Typically, a pharmaceutical composition includes a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, and the like, aswell as from nontoxic organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, aromatic acids, aliphatic and aromatic sulfonic acids and thelike. Base addition salts include those derived from alkaline earthmetals, such as sodium, potassium, magnesium, calcium and the like, aswell as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

The compositions comprising antibody molecules can be formulatedaccording to methods known in the art. Pharmaceutical formulation is awell-established art, and is further described in Gennaro (ed.),Remington: The Science and Practice of Pharmacy, 20^(th) ed.,Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) Ed.,Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); andKibbe (ed.), Handbook of Pharmaceutical Excipients AmericanPharmaceutical Association, 3^(rd) ed. (2000) (ISBN: 091733096X).

Pharmaceutical compositions may be in a variety of forms. These include,for example, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform can depend on the intended mode of administration and therapeuticapplication. Typically, compositions for the agents described herein arein the form of injectable or infusible solutions.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular (IM), intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and by intrasternal injection or byinfusion.

Pharmaceutical compositions may be provided in a sterile injectible form(e.g., a form that is suitable for subcutaneous injection or intravenousinfusion). In some embodiments, pharmaceutical compositions are providedin a liquid dosage form that is suitable for injection or topicalapplication. In some embodiments, pharmaceutical compositions areprovided as in dry form, e.g., as powders (e.g. lyophilized and/orsterilized preparations). The Pharmaceutical composition can be providedunder conditions that enhance stability, e.g., under nitrogen or undervacuum. Dry material can be reconstituted with an aqueous diluent (e.g.,water, buffer, salt solution, etc.) prior to injection.

In one embodiment, the pharmaceutical composition containing an anti-HAantibody is administered intranasally. In another embodiment, thepharmaceutical composition containing an anti-HA antibody isadministered by inhalation, such as by oral or by nasal inhalation.

In embodiments the pharmaceutical composition is suitable for buccal,oral or nasal delivery, e.g., as a liquid, spray, or aerosol, e.g., bytopical application, e.g., by a liquid or drops, or by inhalation). Inembodiments a pharmaceutical preparation comprises a plurality ofparticles, suitable, e.g., for inhaled or aerosol delivery. Inembodiments the mean particle size of 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 microns. In embodiments a pharmaceutical preparation is formulated asa dry powder, suitable, e.g., for inhaled or aerosol delivery. Inembodiments a pharmaceutical preparation is formulated as a wet powder,through inclusion of a wetting agent, e.g., water, saline, or otherliquid of physiological pH. In embodiments a pharmaceutical preparationis provided as drops, suitable, e.g., for delivery to the nasal orbuccal cavity.

In embodiments the pharmaceutical composition is disposed in a deliverydevice, e.g., a syringe, a dropper or dropper bottle, an inhaler, or ametered dose device, e.g., an inhaler.

In one embodiment, a pharmaceutical composition contains a vector, suchas an adenovirus-associated virus (AAV)-based vector, that encodes aheavy chain of an anti-HA antibody molecule, and a light chain of ananti-HA antibody molecule featured in the disclosure. The compositioncontaining the vector can be administered to a subject, such as apatient, such as by injection, e.g., IM injection. Genes encoding theanti-HA antibody under control of, for example, cytomegalovirus (CMV)promoters, are expressed in the body, and the recombinant anti-HAantibody molecule is introduced into the circulation. See, e.g., Balazset al., Nature 30:481:81-84, 2011.

Pharmaceutical compositions typically should be sterile and stable underthe conditions of manufacture and storage. A pharmaceutical compositioncan also be tested to insure it meets regulatory and industry standardsfor administration.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating an agent described herein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating an agent described herein intoa sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,typical methods of preparation are vacuum drying and freeze-drying thatyields a powder of an agent described herein plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

A pharmaceutical composition may be provided, prepared, packaged, and/orsold in bulk, as a single unit dose, and/or as a plurality of singleunit doses. Typically a bulk preparation will contain at least 2, 5, 10,20, 50, or 100 unit doses. A unit dose is typically the amountintroduced into the patient in a single administration. In embodimentsonly a portion of a unit dose is introduced. In embodiments a smallmulitiple, e.g., as much as 1.5, 2, 3, 5, or 10 timies a unit dose isadministered. The amount of the active ingredient is generally equal toa dose which would be administered to a subject and/or a convenientfraction of such a dose such as, for example, one-half or one-third ofsuch a dose.

Immnunogens and Vacccines

Antibodies of the invention have elucidated epitopes that are useful forinducing immunity to, and in embodiments, provide protection from, oneor more, e.g., at least two, influenza strains. These epitopes arereferred to herein as “broad range immunogens.” In an embodiment thebroad range immunogen induces immunity, and in embodiments, confersprotection against at least one Group 1 strain, and a second strainselected from a Group 1 strain, a Group2 strain, and an influenza Bstrain. A broad range immunogen, as the term is used herein, comprises apolypeptide having sufficient sequence and three dimensional structureof an HA, e.g., a HA from a Group 1 strain, e.g., an H1N1 strain, e.g.,A/South Carolina/1/1918, A/Puerto Rico/08/1934, or A/California/04/2009,or an H5N1 strain, e.g., A/Indonesia/5/2005 or A/Vietnam/1203/2004, toallow binding of an antibody of the invention, e.g., one of Ab 044, Ab069, Ab 032 and Ab 031, to the broad range immunogen. In embodiments thebroad range immunogen comprises the epitope of an antibody describedherein e.g., one of Ab 044, Ab 069, Ab 032, and Ab 031. In embodiments,a broad range immunogen does not bind one or more of Ab 67-11, FI6,FI28, C179, or CR6261. In an embodiment Ab 044 binds the broad rangeimmuongen with at least 50, 60, 70, 80, 90, 95, or 99% of the affinitywith which it binds a native HA, e.g., a HA from a Group 1 strain, e.g.,an H1N1 strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. In an embodiment CR6261 binds the broad rangeimmunogen with less than 60, 50, 40, 30, 20, or 10% of the affinity withwhich it binds binds a native HA, e.g., a HA from a Group 1 strain,e.g., an H1N1 strain, e.g., A/South Carolina/1/1918, A/PuertoRico/08/1934, or A/California/04/2009, or an H5N1 strain, e.g.,A/Indonesia/5/2005 or A/Vietnam/1203/2004. In an embodiment the broadrange immunogen differs from wildtype by at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, or 40 residues. In an embodiment the broad rangeimmunogen binds to one or more of Ab 67-11, FI6, FI28, C179, or CR6261with less than 60, 50, 40, 30, 20, or 10% of the affinity with which itbinds binds a native HA, e.g., a HA from a Group 1 strain, e.g., an H1N1strain, e.g., A/South Carolina/1/1918, A/Puerto Rico/08/1934, orA/California/04/2009, or an H5N1 strain, e.g., A/Indonesia/5/2005 orA/Vietnam/1203/2004. In an embodiment the broad range immunogen binds toAb 044 with an affinity that is at least 10, 30, 50, 100, or 200%greater than its affinity for CR626. In an embodiment the epitope of oneof Ab 044, Ab 069, Ab 032, and Ab 031, e.g, Ab 044, is theimmunodominant epitople on the broad range immunogen.

As used herein, the term “broad range vaccine” refers to a preparationcomprising a broad range immunogen, or a nucleic acid endoding a broadrange immunogen, that can induce formation of antibodies or immunityagainst the broad range immunogen or an organism, e.g., an influenzavirus. The broad range immunogen can include dead or weakened virus orantigenic determinants from the organism, e.g., an influenza virus.Typically the broad range vaccine will include one or more additionalcomponents, e.g., carriers, adjuvants and the like.

In an embodiment a broad range vaccine comprises two broad rangeimmunogens, or nucleic acid encoding two broad range immunogens.

A broad range immunogen disclosed here, and vaccines including a broadrange immunogen or a nucleic acid encoding a broad range immunogen(broad range vaccines), can be used to elicit an immune response, in asubject, e.g., a human subject, against one or more influenza virusesdescribed herein. In embodiments the broad range vaccine confersprotection against one or more of the influenza viruses describedherein, e.g., it decreases the chance of developing an infection or thesymptom of an infection, or moderates the severity of an infection.Broad range vaccines of the invention can comprise an HA polypeptidecomprising a broad range immunogen, a nucleic acid encoding a HApolypeptide comprising a broad range immunogen, a particle, e.g., a VLP,liposome, nanoparticle, or microparticle, comprising a broad rangeimmunogen or an nucleic acid that encodes a broad range immunogen.Vaccines can comprise live or inactivated, e.g., replication deficient,viruses. Influenza, as well as other viruses can be used in broad rangevaccines.

As used herein, the term “immunogen” or “antigenic formulation” or“antigenic composition” refers to a preparation which, when administeredto a vertebrate, e.g., a mammal, e.g., a human, can induce an immuneresponse.

Vaccine Formulation

A broad range immunogen, e.g., a polypeptide, or VLP, liposome,nanoparticle, or microparticle comprising a broad range immunogen, canbe formulated into compositions that further comprise a pharmaceuticallyacceptable carrier or excipient. A nucleic acid that encodes a broadrange immunogen can be formulated into a composition that furthercomprises a pharmaceutically acceptable carrier or excipient. A carrieror excipient is a pharmaceutical agent that does not itself induce theproduction of an immune response harmful to the animal receiving thecomposition and which may be administered as a vaccine component withoutcausing undue toxicity. As used herein, the term “pharmaceuticallyacceptable vaccine component” includes components, e.g., a carrier, thathave been approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopia, European Pharmacopia, orother generally recognized pharmacopia for use in mammals, e.g., inhumans. Non-limiting examples of pharmaceutically acceptable carriersare saline, buffered saline, dextrose, water, glycerol, sterile isotonicaqueous buffer, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate, and combinations thereof. Inone embodiment, the formulation can be used for administration of thevaccine to humans. In some embodiments, the formulation is sterile, freefrom particulate matter, and/or non-pyrogenic. The vaccine may alsoinclude one or more of: a wetting agent, an emulsifying agent, and abuffering agent. The vaccine can be in solid form, e.g., a lyophilizedpowder, liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder.

In some embodiments, broad range vaccines may include one or moreadjuvants. Adjuvants are agents that enhance immune responses, and theiruse is known in the art (see, e.g., “Vaccine Design: The Subunit andAdjuvant Approach”, Pharmaceutical Biotechnology, Volume 6, Eds. Powelland Newman, Plenum Press, New York and London, 1995). Non-limitingexamples of adjuvants are complete Freund's adjuvant (CFA), incompleteFreund's adjuvant (IFA), squalene, squalane, aluminum hydroxide,aluminum salts, calcium salts, and saponin fractions derived from thebark of the South American tree Quillaja Saponaria Molina (e.g., QS21).In some embodiments the adjuvant may be an emulsion comprising oil andwater. The oil phase may comprise squalene, squalane, and/or asurfactant. The surfactant may be a non-ionic surfactant, e.g., a mono-or di-Ci2-C24-fatty acid ester of sorbitan or mannide

Synthetic variants of molecules recognized by Toll-Like Receptors (TLRs)may also be used as adjuvants. TLRs help the body to distinguish betweenself and non-self molecules by recognizing molecular patterns associatedwith pathogens.

Molecules recognized by TLRs include double-stranded RNA,lipopolysaccharides, single-stranded RNA with viral-specific orbacterial-specific modifications, and DNA with viral-specific orbacterial-specific modifications. Synthetic molecules that mimic theproperties of these naturally-occurring molecules recognized by TLRshelp to trigger an immune response and therefore can be used asadjuvants. Non-limiting examples of such synthetic molecules includepolyriboinosinic:polyribocytidylic aci (poly (I:C)), double-strandednucleic acids with at least one locked nucleic acid nucleoside,attenuated lipid A derivatives (ALDs) (e.g., monophosphoryl lipid A and3-deacyl monophosphoryl lipid A), and imiquimod.

Vaccines may be formulated with or administered in combination with theadministration of immune stimulators Immune stimulators are moleculesthat increase the response of the immune system. Non-limiting examplesof immune stimulators are cytokines, lymphokines, and chemokines thathave immunostimulatory, immunopotentiating, and pro-inflammatoryactivities, such as interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-12,IL-13), growth factors (e.g., granulocyte-macrophage (GM)-colonystimulating factor (CSF)); and other immunostimulatory molecules, suchas macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc Immunestimulators may be administered in the same formulation as the VLPs ormay be administered separately Immune stimulators may be administered asproteins or as nucleic acids from which the immunostimulatory proteincan be expressed.

Administration

Generally, broad range vaccines will be administered in an effectiveamount or quantity sufficient to stimulate an immune response againstone or more strains of influenza. Vaccine dosage can be adjusted withinthis range based on clinical factors, e.g., age, physical condition,body weight, sex, diet, and time of administration.

Methods of administering a broad range immunogen or broad range vaccineinclude enteral and parenteral administration. They can also be providedby epidural or mucosal administration (e.g., intranasal and oral orpulmonary routes or by suppositories). They can be provided byinhalation or direct contact with the buccal or nasal cavities. Inembodiments a broad range immunogen or broad range vaccine isadministered intramuscularly, intravenously, subcutaneously,transdermally, or intradermally. The compositions may be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra,urinary bladder and intestinal mucosa, etc.) and by any convenientmeans, for example, by injection using a needle and syringe or aneedleless injection device, by drops, via an aerosol comprising largeparticles, or by spray into the upper respiratory tract. A broad rangeimmunogen or vaccine may be administered together with otherbiologically active agents, for example, immunogenic agents, forexample, antivirals and/or antibiotics.

In some embodiments a broad range immunogen or broad range vaccine isadministered so as to target mucosal tissues in order to elicit animmune response at the site of immunization. For example, mucosaltissues can be targeted for immunization by using oral administration ofcompositions which contain adjuvants with particular mucosal targetingproperties. Examples of mucosal tissues that can be targeted include,but are not limited to, gut-associated lymphoid tissue (GALT),nasopharyngeal lymphoid tissue (NALT), and bronchial-associated lymphoidtissue (BALT).

A broad range immunogen or broad range vaccine may be administered on adosage schedule, for example, by sequential administrations to subject.In some embodiments a first dose of the composition is followed after aperiod of time by a second dose. The period of time between the firstand second doses may be anywhere from two weeks to one year, forexample, about 1, about 2, about 3, about 4, about 5 to about 6 months.In some embodiments the second dose of the composition is followed by athird dose administered a period of time after the first dose. Theperiod of time between the first and third doses may be anywhere fromabout three months to about two years or more, for example, about 4,about 5, or about 6 months, or about 7 months to about one year. In someembodiments the second, third, or higher dose is administered when thelevels of specific immunoglobulins in the serum, urine, and/or mucosalsecretions of the subject drop below a threshold. In one embodiment theperiod of time between the first and second doses is about one month andthe period of time between the first and third doses is about sixmonths. In another embodiment the period of time between the first andsecond doses is about six months. In some embodiments, for example, whenthe subject is a neonate or infant, doses can be administered throughoutchildhood. Other factors that put the subject at increased risk ofinfection, for example, subjects who are health care workers, day careworkers, family members of young children, the elderly, and/orindividuals with compromised cardiopulmonary function, may influence thedosage schedule, for example, may require the subject to have more dosesor more frequent doses. When multiple doses are required, the doses maybe administered by the same or different routes.

One skilled in the art can readily determine the dosage of the broadrange immunogen or broad range vaccine. For example, the dosage may bedetermined by identifying doses that elicit a protective or therapeuticimmune response, for example, by measuring the level of specificimmunoglobulins in the serum or measuring the inhibitory ratio ofantibodies in samples of serum, urine, or mucosal secretions from asubject. Dosages can be determined from studies in animals, for example,in guinea pigs, hamsters, ferrets, chinchillas, mice, or rats. An animalneed not be a natural host to a particular infectious agent to serve asa subject in studies of the disease caused by said infection agent.Dosages can also be determined from clinical studies in humans, whichare routine in the art. The skilled artisan will understand that theroute of administration will affect the dosage. Dosages can also becalculated from dose-response curves obtained from in vitro studies orstudies using animal models.

A broad range immunogen or vaccine can be administered a subject thatdoes not have a disease caused by influenza virus infection or has notbeen and is not currently infected with an influenza virus infection,e.g., a broad range immunogen or vaccine can be administered to asubject at risk for infection. A broad range immunogen or vaccine can beadministered to a subject infected with a first influenza strain, e.g.,to protect against infection with a second strain. In embodiments thebroad range immunogen or vaccine is protective against the first strain.In embodiments the broad range immunogen or vaccine is not protectiveagainst the first strain.

In embodiments the subject is an adult, an adult over 50 years of age, aperson less than 18 years of age, a person less than 2 years of age, ora person less than 6 months of age.

In an embodiment the subject is at risk for a disorder of the lung,e.g., cystic fibrosis, emphysema, asthma, or bacterial infections, orcardiovascular disease. In an embodiment the subject isimmune-compromised. In an embodiment the subject is a health careprovider, e.g., a physician, nurse, or aid. In an embodiment the subjectworks at or regularly visits, or lives in a hospital, nursing home,assisted care facility, clinic, or doctor's office.

Broad range immunogens and broad range vaccines can be administeredeither alone or in combination with one or more other therapy or agent,e.g., the administration of a second or additional agent, e.g., toprevent or delay or minimize one or more symptoms or effects of aninfluenza infection.

In embodiments the combination can result in a lower dose of the broadrange vaccine or of the other therapy being needed, which, inembodiments can reduce side effects. In embodiments the combination canresult in enhanced delivery or efficacy of one or both agents. Theagents or therapies can be administered at the same time (e.g., as asingle formulation that is administered to a patient or as two separateformulations administered concurrently) or sequentially in any order.

Such second or additional agents include other vaccines, anti-viralagents, and/or antibodies. In typical embodiments the second oradditional agent is not co-formulated with the binding agent, e.g.,antibody molecule, though in others it is.

In embodiments the broad range vaccine and the second or additionalagent are administered such that one or more of the following isachieved: therapeutic levels, or therapeutic effects, of one overlap theother; detectable levels of both are present at the same time; or thetherapeutic effect is greater than what would be seen in the absence ofeither the broad range vaccine, or the second or additional agent. Inembodiments each agent will be administered at a dose and on a timeschedule determined for that agent.

The second or additional agent can be, for example, for treatment orprevention of influenza. For example, a broad range vaccine providedherein can be administered in combination with another vaccine, e.g., amixture (a.k.a. a cocktail) of influenza peptides to stimulate thepatient's immune system to prevent infection with particular strains ofinfluenza A. In other examples, the second or additional agent is ananti-viral agent (e.g., an anti-NA or anti-M2 agent), a pain reliever,an anti-inflammatory, an antibiotic, a steroidal agent, a secondtherapeutic antibody molecule (e.g., an anti-HA antibody), an adjuvant,a protease or glycosidase (e.g., sialidase), etc.

Exemplary anti-viral agents include, e.g., vaccines, neuraminidaseinhibitors or nucleoside analogs. Exemplary anti-viral agents caninclude, e.g., zidovudine, gangcyclovir, vidarabine, idoxuridine,trifluridine, foscarnet, acyclovir, ribavirin, amantadine, remantidine,saquinavir, indinavir, ritonavir, alpha-interferons and otherinterferons, a neuraminidase inhibitor (e.g., zanamivir (Relenza®),oseltamivir (Tamiflu®), laninamivir, peramivir), rimantadine. Exemplaryantibody molecules include, for example, Ab 67-11 (U.S. Provisionalapplication No. 61/645,453, FI6 (U.S. Published Application No.2010/0080813), FI28 (U.S. Published Application No. 2010/0080813), C179(Okuno et al., J. Virol. 67:2552-8, 1993), F10 (Sui et al., Nat. Struct.Mol. Biol. 16:265, 2009), CR9114 (Dreyfus et al., Science 337:1343,2012), or CR6261 (see, e.g., Ekiert et al., Science 324:246, 2009).Other exemplary antibodies include those described herein, e.g., Ab 044,Ab 069, Ab 032, or Ab 031. In the case of combinations, two agents canbe administered as part of the same dosage unit or administeredseparately. Other exemplary agents useful for treating the symptomsassociated with influenza infection are acetaminophen, ibuprofen,aspirin, and naproxen.

In an embodiment the broad range vaccine and the second or additionalagent are provided as separate formulations, and the step ofadministering includes sequentially administering the broad rangevaccine and the second or additional agent. The sequentialadministrations can be provided on the same day (e.g., within one hourof one another or at least 3, 6, or 12 hours apart) or on differentdays.

In embodiments the broad range vaccine and the second or additionalagent are each administered as a plurality of doses separated in time.The broad range vaccine and the second or additional agent are generallyeach administered according to a regimen. The regimen for one or bothmay have a regular periodicity. The regimen for the broad range vaccinecan have a different periodicity from the regimen for the second oradditional agent, e.g., one can be administered more frequently than theother. In one implementation, one of the broad range vaccine and thesecond or additional agent is administered once weekly and the otheronce monthly. In another implementation, one of the broad range vaccineand the second or additional agent is administered continuously, e.g.,over a period of more than 30 minutes but less than 1, 2, 4, or 12hours, and the other is administered as a bolus. In embodimentssequential administrations are administered. The time betweenadministration of the one agent and another agent can be minutes, hours,days, or weeks. The use of a broad range vaccine described herein canalso be used to reduce the dosage of another therapy, e.g., to reducethe side-effects associated with another agent that is beingadministered. Accordingly, a combination can include administering asecond or additional agent at a dosage at least 10, 20, 30, or 50% lowerthan would be used in the absence of the broad range vaccine. The broadrange vaccine and the second or additional agent can be administered byany appropriate method, e.g., subcutaneously, intramuscularly, orintravenously.

In some embodiments, each of the broad range vaccine and the second oradditional agent is administered at the same dose as each is prescribedfor monotherapy. In other embodiments, the broad range vaccine isadministered at a dosage that is equal to or less than an amountrequired for efficacy if administered alone. Likewise, the second oradditional agent can be administered at a dosage that is equal to orless than an amount required for efficacy if administered alone.

In some cases, the formulations described herein, e.g., formulationscontaining an broad range vaccine featured in the disclosure, includeone or more second or second or additional agents, e.g., a second oradditional agent, or are administered in combination with a formulationcontaining one or more second or additional agents.

In an embodiment, administration of a broad range vaccine is by one ofthe following routes: oral, intravenous, intramuscular, intra-arterial,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by liquids, powders,ointments, creams, sprays, or drops), mucosal, nasal, buccal, enteral,sublingual; intratracheal instillation, bronchial instillation, and/orinhalation; and/or as an oral spray, nasal spray, and/or aerosol.

In an embodiment a broad range vaccine is administered by inhalation oraerosol delivery of a plurality of particles, e.g., particles comprisinga mean particle size of 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 microns.

Vaccines of the invention can be combined with the secondary agents andtreatments discussed in the context of treatment with antibodies of theinvention elsewhere herein.

Virus-Like Particles

A broad range immunogen can be provided in a virus-like particle (VLP).A VLP is a structure that shares some component and structuralsimilarity to a virus but generally is not infectious. VLPs typicallylack a viral genome and therefore cannot reproduce. VLPs can be producedby cloning and co-expressing one or more viral proteins, typicallyincluding an antigenic protein of interest, in a cell, and recoveringfrom the cells VLPs that include the antigenic protein of interest. Thisis described in more detail below.

Cloning

Methods of molecular cloning are known in the art (see, e.g., Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152 Academic Press, Inc., San Diego, Calif., and Sambrook et al,Molecular Cloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2000). Methods of molecularcloning include techniques for polypeptide engineering and mutagenesis,which allow for the deletion, insertion, substitution, and otheralterations of amino acids within a polypeptide. Methods of molecularcloning also include techniques for isolation and manipulation ofnucleic acids that encode polypeptides or that increase, decrease,regulate, or otherwise alter the expression of polypeptides. Methods ofmolecular cloning further comprise vectors that facilitate the geneticmanipulation and expression of polypeptides and nucleic acids.

A vector is a vehicle through which a nucleic acid can be reproduced ortransmitted between cells or living organisms. A vector may be, but isnot limited to, a plasmid, virus, bacteriophage, provirus, phagemid,transposon, or artificial chromosome. A vector may replicateautonomously or via the machinery of a host cell or organism. A vectormay comprise DNA and/or RNA, which may exist in an isolated form or in acomplex with other components, e.g., proteins. Molecular cloning methodsmay be used to insert exogenous nucleic acids into vectors to createconstructs for the expression of nucleic acids and/or polypetpides,e.g., influenza HA.

Expression

Methods of expressing exogenous nucleic acids and/or polypeptides areknown in the art (see, e.g., Sambrook). Typically, exogenous expressionentails introducing an expression construct, created using the methodsdescribed above, into a host cell or organism and allowing thebiochemical machinery of the host cell to produce one or more of theforeign nucleic acids and/or polypeptides. The host cell may be, but isnot limited to, a prokaryotic cell, e.g., a bacterium, or a eukaryoticcell, e.g., a fungal, plant, avian, amphibian, nematode, insect, ormammalian, e.g., a mouse, hamster, monkey, or human, cell. Examples ofinsect cells include Sf9, SfZ1, High Five cells, and Drosophila S2cells. Examples of fungi (including yeast) host cells are S. cerevisiae,Kluyveromyces lactis, species of Candida albicans, Candida glabrata,Aspergillus nidulans, Schizosaccharomyces pombe, Pichia pastoris, andYarrowia lipolytica. Examples of mammalian cells include COS cells, babyhamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary(CHO) cells, human embryonic kidney (HEK) cells, African green monkeycells, CV1 cells, HeLa cells, MDCK cells, Vero, and Hep-2 cells. Anexample of an amphibian cell is an oocyte from Xenopus laevis. Examplesof prokaryotic cells include E. coli, B. subtilis, and mycobacteria. Thehost cell may be part of a multicellular organism or may be grown invitro, e.g., in a culture of a tissue, organ, a mixed population ofcells, or a clonal population of cells. The expression construct may beintroduced into the host cell by, e.g., transfection, transduction,transformation, electroporation, microinjection, lipofection, orinfection.

VLPs can be produced by culturing host cells into which one or moreconstructs that enable the expression of exogenous polypeptides havebeen introduced. The exogenous proteins may be polypeptides identical toor derived from the polypeptides of the influenza virus, e.g., M1, HA,or NA, fragments of M1, HA, or NA, or variants of M1, HA, or NA. Theexpression construct may contain one or more additional elements, e.g.,a marker, e.g., a selectable marker, or an origin of replication.Methods to grow cells for production of VLPs include, but are notlimited to, batch, batch-fed, continuous and perfusion cell culturetechniques. Methods and reagents may be used to increase efficiency ofVLP production. For example, a leader sequence, e.g., a signal sequence,may be added to one or more exogenous polypeptides, e.g., M1, HA, and/orNA, to facilitate transport of the exogenous polypeptide(s) within thehost cell.

Isolation and Purification of VLPs

VLPs can be isolated and purified using methods known in the art, suchas density gradient centrifugation, filtration, ion exchangechromatography, and gel filtration chromatography. Using the methodsdescribed above, VLPs are produced by host cells and secreted into theculture medium. A typical stepwise procedure for isolating and purifyingVLPs from the culture medium involves (1) ultrafiltration of the culturemedium to concentrate VLPs, (2) diafiltration of VLPs to removecomponents of the culture medium, (3) centrifugation of VLPs on asucrose density gradient to remove cellular debris and particulatematter, and (4) anion exchange chromatography of VLPs to remove nucleicacids.

Vesicles

A broad range immunogen may be incorporated into, or packaged in, avesicle.

Typically, vesicles have an aqueous compartment enclosed by one or morebilayers comprising amphipathic molecules (e.g., fatty acids, lipids,steroids, etc.). A broad range immunogen may be contained within theaqueous core of the vesicle or may be localized to the amphipathicbilayer.

In some embodiments the amphipathic molecules of the vesicle arenonionic, e.g., a nonionic surfactant. For example, the nonionicamphipathic molecule may be a glyercol-based, ester-linked surfactant.Such glycerol esters may comprise one of two higher aliphatic acylgroups, e.g., an acyl group containing at least ten carbon atoms in eachacyl moiety. Surfactants based on such glycerol esters may comprise morethan one glycerol unit, e.g., 2, 3, 4, or 5 glycerol units. Glycerolmonoesters may be used, e.g., those containing a C12-C20 alkanoyl oralkenoyl moiety, for example caproyl, lauroyl, myristoyl, palmitoyl,oleyl or stearoyl. An exemplary ester linked surfactant based onglycerol is 1-monopalmitoyl glycerol.

The nonionic amphipathic molecule of the vesicle bilayer may also be anether-linked surfactant. For example, ether-linked surfactants based onglycerol or a glycol having a lower aliphatic glycol of up to 4 carbonatoms, such as ethylene glycol, may be used. Surfactants based on suchglycols may comprise more than one glycol unit, e.g., 2, 3, 4, or 5glycol units (e.g., diglycolcetyl ether and/or polyoxyethylene-3-laurylether). Glycol or glycerol monoethers may be used, including thosecontaining a C12-C20 alkanyl or alkenyl moiety, for example capryl,lauryl, myristyl, cetyl, oleyl or stearyl. For examples of ethyleneoxide condensation products that can be used as amphipathic molecules,see PCT Publication No. WO88/06882 (e.g., polyoxyethylene higheraliphatic ether and amine surfactants). Non-limiting examples ofether-linked surfactants are 1-monocetyl glycerol ether anddiglycolcetyl ether.

In some embodiments the vesicle comprising a nonionic surfactant mayalso comprise an ionic amphipathic molecule. For example, an ionicamphiphile may cause the vesicles become negatively charged, which mayhelp stabilize the vesicles and promote dispersion. Ionic amphipathicmolecules that can be incorporated into vesicles include, but are notlimited to, higher alkanoic and alkenoic acids (e.g., palmitic acid,oleic acid) and other compounds containing acidic groups, for example,phosphates, (e.g., dialkyl phosphates, e.g., dicetylphosphate, orphosphatidic acid or phosphatidyl serine) and sulphate monoesters (e.g.,higher alkyl sulphates, e.g., cetylsulphate). The ionic amphiphile maybe present at between 1% and 30%, between 2% and 20%, or between 5% and15% the amount, by weight, of nonionic surfactant.

In some embodiments the vesicle may further comprise a high-molecularweight hydrophobic molecule capable of forming a bilayer, for example, asteroid, for example, cholesterol. The presence of the steroid mayfacilitate formation of the bilayer, for example, by conferring physicalproperties on the bilayer. The steroid may be present at between 20% and120%, between 25% and 90%, or between 35% and 75% amount, by weight, ofnonionic surfactant

In some embodiments, the vesicle may be a bilosome (see, e.g., U.S. Pat.No. 5,876,721). As used herein, “bilosomes” are vesicles that comprisenon-ionic surfactants and transport enhancing molecules which facilitatethe transport of lipid-like molecules across mucosal membranes.

Methods for preparing vesicles comprising nonionic surfactants are knownin the art. The skilled artisan will understand that such methods may beused to prepare vesicles comprising a broad range immunogen.

Viral Vectors

A broad range immunogen can be provided in an influenza virus. In anembodiment the broad range immunogen is incorporated in an HApolypeptide, e.g., an HA polypeptide that differs from wildtype. Inembodiments the HA polypeptide that comprises the broad range immunogenis other than a wild type sequence, e.g., an engineered sequence. It canbe incorporated into a virion by supplying the polypeptide in transduring production of the virion or the genome of the virus can beengineered to produce it. In either case, viral particles comprising thebroad range immunogen are produced. In an embodiment the virus isengineered to have an attenuated phenotype, e.g., the virus can have no,or only very low levels of, replication in human cells. In embodimentsthe virus is inactivated. Inactivation methods include contact withdenaturants, e.g., formalin, heat, or detergent. A broad range immunogencan be provided in a non-influenza virus, e.g., the non-influenza virusvector can be a Newcastle disease virus, a vaccinia virus, anadenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus.

Kits

In one embodiment, the broad range immunogen or vaccine is packaged in akit. In some embodiments, the kit comprises two containers, one of whichcontains the broad range immunogen and the other of which contains anadjuvant. In some embodiments the kit comprises two containers, one ofwhich contains broad range immunogen as a lyophilized powder and theother of which contains a liquid for resuspending the broad rangeimmunogen. The kit may contain a notice as required by governmentalagency regulating the manufacture, use, and sale of pharmaceuticals orbiological products, the notice indicating that the composition has beenapproved for manufacture, use, and/or sale for administration to humans

The vaccine may be supplied in a hermetically-sealed container. Thevaccine may be provided as a liquid or as a lyophilized powder that canbe reconstituted by the addition, e.g., of water or saline, to aconcentration suitable for administration to a subject.

Epitope

HAs exist in nature as homotrimers of proteolytically processed maturesubunits. Each subunit of the trimer is synthesized as a precursor. Aprecursor molecule is proteolytically processed into two disulfidebonded polypeptide chains to form a mature HA polypeptide. The mature HApolypeptide includes two domains: (1) a core HA-1 domain that extendsfrom the base of the molecule through the fibrous stem to the membranedistal head region that contains the glycan receptor binding domain,returning to fibrous region ending in the cleavage site, and (2) HA-2domain that includes the stem region and the transmembrane domain of HA.HA-1 includes a glycan binding site. The glycan binding site may beresponsible for mediating binding of HA to the HA-receptor. The HA-2domain acts to present the HA-1 domain. The HA trimer can be stabilizedby polar and non-polar interactions between the three long HAalpha-helices of the stem of HA monomers.

HA sequences from all influenza subtypes share a set of amino acids inthe interface of the HA-1 and HA-2 domains that are well conserved. TheHA-1/HA-2 interface membrane proximal epitope region (MPER) thatincludes the canonical α-helix and residues in its vicinity are alsoconserved across a broad spectrum of subtypes. (Ekiert et al., Science,324(5924):246, 2009; Sui et al., Nat Struct Mol Biol. 16(3):265, 2009).

Ab 044 has high affinity for HA's from Group 1 and Group 2. It binds aconformational epitope that is broadly conserved across a plurality ofinfluenza strains. Numerous amino acid residues distributed along thelinear sequences of HA from different strains/subtypes contribute the Ab044 conformational epitope. The interaction of Ab044 with H3 wasanalyzed by docking studies and residues bound by (or not bound by)Ab044 were identified.

The Fv of Ab 044 was docked against HA of group I and II strains usingZDOCK. The structure of the HA antigen was modeled using the SWISS MODELhomology modeling server keeping the solved crystal structure of H1N1 asthe template. ZDOCK uses shape complementarity along with desolvationand electrostatic energy terms (‘ZRANK’) to rank docked poses. To ensurethe docked poses do not deviate significantly from the native complex,mapped epitope and paratope residues by alanine scanning are forced tobe included in the binding interface.

For comparison studies, amino acids that bind (or do not bind) FI6 weretaken from published US patent application US 2011/0274702 A1,Neutralizing Anti-Influenza A Virus Antibodies and Uses Thereof, filedJul. 18, 2011.

ZDOCK is a Fast Fourier Transform based protein docking program. It wasdeveloped by Zhiping Weng at the University of Massachusetts MedicalSchool. In ZDOCK, two PDB files are input and the output is thepredicted structure of their complex. The program searches all possiblebinding modes in the translational and rotational space between the twoproteins and evaluates each by an energy scoring function. The protein'sstructure is converted to a digital signal and a Fast Fourier Transformtechnique used to reduce computational time. ZDOCK is discussed inPierce B G, Hourai Y, Weng Z. (2011) Accelerating Protein Docking inZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9): e24657,Pierce B, Tong W, Weng Z. (2005) M-ZDOCK: A Grid-based Approach forC_(n) Symmetric Multimer Docking. Bioinformatics 21(8): 1472-1476;Mintseris J, Pierce B, Wiehe K, Anderson R, Chen R, Weng Z. (2007)Integrating Statistical Pair Potentials into Protein Complex Prediction.Proteins 69(3): 511-520; and Chen R, Li L, Weng Z. (2003) ZDOCK: AnInitial-stage Protein Docking Algorithm. Proteins 52(1): 80-7.

SWISS-MODEL is a fully automated protein structure homology-modelingserver. It is accessible via the ExPASy web server, or from the programDeepView (Swiss Pdb-Viewer). Swiss-Model is discussed in Arnold K.,Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: Aweb-based environment for protein structure homology modelling.Bioinformatics, 22, 195-201; Kiefer F, Arnold K, Kunzli M, Bordoli L,Schwede T (2009). The SWISS-MODEL Repository and associated resources.Nucleic Acids Research. 37, D387-D392; and Peitsch, M. C. (1995) Proteinmodeling by E-mail Bio/Technology 13: 658-660.

H3 residues that bind Ab 044 and H3 residues that bind FI6 are discussedbelow.

H3 HA1

The amino acid sequence of H3 HA1 is provided below, as SEQ ID NO: 173.Residues N38, I278, and D291 shown in dashed boxes, are bound by Ab 044but not by FI6; Residues Q327, T328, and R329 shown in dotted boxes, arebound by FI6 but not by Ab 044; residues T318, R321, and V323 shown insolid boxes, are bound by both Ab 044 and FI6.

(SEQ ID NO: 173)

ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVER SKAFSNCYPY DVPDYASLRSLVASSGTLEF ITEGFTWTGV TQNGGSNACK RGPGSGFFSR LNWLTKSGST YPVLNVTMPNNDNFDKLYIW GIHHPSTNQE QTSLYVQASG RVTVSTRRSQ QTIIPNIGSR PWVRGLSSRI

H3 HA2

The amino acid sequence of H3 HA21 is provided below, as SEQ ID NO: 174Residue N12 shown in a dash box, is bound by Ab 044 but not by FI6;Residues G1, L2, F3, G4, and D46 shown in dotted boxes, are bound by FI6but not by Ab 044; residues A7, E11, I18, D19, G20, W21, L38, K39, T41,Q42, A43, I45, I48, N49, L52, N53, I56, and E57, shown in solid boxes,are bound by both Ab 044 and FI6.

(SEQ ID NO: 174)

TDSEMNKLFE KTRRQLRENA EEMGNGCFKI YHKCDNACIE SIRNGTYDHD VYRDEALNNR FQIKG 

H1 residues that bind Ab 044 and H1 residues that bind FI6 are discussedbelow.

H1 HA1

The amino acid sequence of H1 HA1 is provided below, as SEQ ID NO: 181.Residues H31, N279, and 5292 shown in dashed boxes, are bound by Ab 044but not to by FI6. Residues Q328 and 5329 shown in dotted boxes, arebound by FI6 but not by Ab 044. Residues T319, R322, and I324 shown insolid boxes, are bound by both Ab 044 and FI6.

(SEQ ID NO: 181)

EDSHNGKLCK LKGIAPLQLG KCNIAGWLLG NPECDLLLTA SSWSYIVETS NSENGTCYPG DFIDYEELRE QLSSVSSFEK FEIFPKTSSW PNHETTKGVT AACSYAGASS FYRNLLWLTK KGSSYPKLSK SYVNNKGKEV LVLWGVHHPP TGTDQQSLYQNADAYVSVGS SKYNRRFTPE IAARPKVRDQ AGRMNYYWTLLEPGDTITFE ATGNLIAPWY AFALNRGSGS GIITSDAPVH

H1 HA2

The amino acid sequence of H1 HA2 is provided below, as SEQ ID NO: 182.Residues G12 shown in a dashed box, is bound by Ab 044 but not by FI6.Residues G1, L2, F3, G4, and D46 shown in dotted boxes, are bound by FI6but not by Ab 044. Residues A7, E11, I18, D19, G20, W21, Q38, K39, T41,Q42, N43, I45, I48, T49, V52, N53, I56, and E57 shown in solid boxes,are bound by both Ab 044 and FI6.

(SEQ ID NO: 182)

LNKKVDDGFL DIWTYNAELL VLLENERTLD FHDSNVRNLY EKVKSQLKNN AKEIGNGCFE FYHKCDDACM ESVRNGTYDY PKYSEESKLN REEIDGVKLE SMGVYQILAI YSTVASSLVL LVSLGAISFW MCSNGSLQCR ICI 

FIG. 26 is a three dimensional representation of H3 HA with the aminoacids residues that are predicted to be part of Ab044 epitope but notpart of FI6's epitope highlighted. That is, the highlighted amino acidsare unique to Ab044's epitope.

FIG. 27 is a three dimensional representation of H3 HA with the aminoacid residues that are part of FI6's epitope but not predicted to bepart of Ab044's epitope highlighted. indicated.

Diagnostic Methods

The binding agents, e.g., antibody molecules, provided herein are usefulfor identifying the presence of influenza in a biological sample, e.g.,a patient sample, such as a fluid sample, e.g., a blood, serum, saliva,mucous, or urine sample, or a tissue sample, such as a biopsy.

In one embodiment, a patient sample is contacted with a binding agent,e.g., an antibody molecule, featured in the disclosure, and binding isdetected. Binding can be detected with a number of formats and means ofdetection, e.g., with an antigen capture assay, such as an ELISA assayor Western blot, or an immunohistochemistry assay. In embodiments thebinding agent, e.g., an antibody molecule, is provided, e.g., coupled toan insoluble matrix, e.g., a bead or other substrate, and a detectionmolecule used to detect binding of HA.

Binding of binding agent, e.g, antibody molecule, to HA, can be detectedwith a reagent comprising a detectable moiety, e.g., a reagent, e.g., anantibody, which binds the binding agent, e.g., antibody molecule. Inembodiments the binding agent, e.g, antibody molecule, has a detectablemoiety. Suitable detectable moieties include enzymes (e.g., horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase,acetylcholinesterase, glucose oxidase and the like), radiolabels (e.g.,³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), haptens, fluorescentlabels (e.g., FITC, rhodamine, lanthanide phosphors, fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike), phosphorescent molecules, chemiluminescent molecules,chromophores, luminescent molecules, photoaffinity molecules, coloredparticles or affinity ligands, such as biotin, predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, or binding sites for secondary antibodies, metal bindingdomains, epitope tags). In some embodiments, labels are attached byspacer arms of various lengths to reduce potential steric hindrance.

In embodiments, a human is tested for presence of influenza virus be amethod described herein, and if the test is positive, a binding agents,e.g., antibody molecules, e.g., an antibody, provided herein, isadministered.

The binding agents, e.g., antibody molecules, e.g., an antibody,provided herein can be used for cytology assays, such as to identify anHA in a cell. The assay can be a colorimetric assay. A biological samplefrom a normal (non-infected) individual is used as a control. Thediagnostic assay can be performed in vitro.

The diagnostic assay can also be performed to determine infection ofcells in culture, e.g., of mammalian cells in culture. The antibodymolecules can be used in in vitro assays.

Because the antibody molecules featured herein bind a broad spectrum ofHA subtypes, the diagnostic assays featured in the disclosure can detectthe presence of influenza virus in patients infected with a variety ofdistinct strains of influenza. A patient sample can be further testedwith subtype specific antibodies, or other assays (e.g., RFLP(Restriction Fragment Length Polymorphism), PCR (Polymerase ChainReaction), RT-PCR (Reverse Transcription coupled to Polymerase ChainReaction), Northern blot, Southern blot or DNA sequencing) to furtherdetermine the particular strain of virus.

In one embodiment, a patient determined to be infected with influenza Acan be further administered an antibody molecule featured in thedisclosure, to treat the infection.

Also provided are solid substrates, e.g., beads, dipsticks, arrays, andthe like, on which is disposed a binding agent, e.g., antibody molecule.

Kits

A binding agent, e.g., an antibody molecule, disclosed herein, e.g.,generated by the methods described herein, can be provided in a kit. Thekit can inlcuide one or more other compoentents, e.g., containers,buffers or other diluents, deleviery devices, and the like.

In one embodiment, the kit includes materials for administering anantibody molecule to a subject, such as for treatment or prevention ofinfection by influenza viruses. For example, the kit can include one ormore or all of: (a) a container that contains a composition thatincludes an antibody molecule, optionally (b) a container that containsa composition that includes a second therapeutic agent, and optionally(c) informational material.

In another embodiment, the kit includes materials for using an antibodymolecule in a diagnostic assay, such as for detection of HA in abiological sample.

For example, the kit can include one or more or all of: (a) a containerthat contains a composition that includes an antibody molecule,optionally (b) a container that contains a reagents, e.g., labeled witha detectable moiety, to detect the antibody, e.g., for use in an ELISAor immunohistochemistry assay, and optionally (c) informationalmaterial. In other embodiments, the kit comprises a binding agent, e.g.,antibody molecule, comprising a detectable moiety.

In an embodiment, the kit comprises a solid substrate, e.g., bead,dipstick, array, and the like, on which is disposed a binding agent,e.g., antibody molecule.

The informational material can be descriptive, instructional, marketingor other material that relates to the methods described herein and/orthe use of the agents for therapeutic benefit, or for a diagnosticassay.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the antibody, concentration, date of expiration, batch orproduction site information, and so forth. In one embodiment, theinformational material relates to methods of administering the antibody,e.g., in a suitable dose, dosage form, or mode of administration (e.g.,a dose, dosage form, or mode of administration described herein), totreat a subject who has an infection, e.g., viral infection or secondaryinfection (e.g., secondary bacterial infection).

In another embodiment, the informational material relates to methods forusing the antibody molecule for a diagnostic assay, e.g., to detect thepresence of influenza viruses in a biological sample.

The information can be provided in a variety of formats, includingprinted text, computer readable material, video recording, or audiorecording, or information that provides a link or address to substantivematerial.

In addition to the agent, the composition in the kit can include otheringredients, such as a solvent or buffer, a stabilizer, or apreservative. The agent can be provided in any form, e.g., a liquid,dried or lyophilized form, and substantially pure and/or sterile. Whenthe agents are provided in a liquid solution, the liquid solutiontypically is an aqueous solution. When the agents are provided as adried form, reconstitution generally is by the addition of a suitablesolvent. The solvent, e.g., sterile water or buffer, can optionally beprovided in the kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the antibody molecule and the second oradditional agent, such as in a desired ratio. For example, the kit caninclude a plurality of syringes, ampoules, foil packets, blister packs,or medical devices each containing, for example, a single combinationunit dose. The containers of the kits can be air tight, waterproof(e.g., impermeable to changes in moisture or evaporation), and/orlight-tight.

The kit optionally includes a device suitable for administering thecomposition, e.g., a syringe or device for delivering particles oraerosols, e.g., an inhaler, a spray device, or a dropper or othersuitable delivery device. The device can be provided pre-loaded with oneor both of the agents or can be empty but suitable for loading.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

TABLE 4C Nucleic acid and amino acid sequences SEQ ID Lab NO. no. SourceComment   1 n.a. Table 2 Consensus AA sequence of HC CDR1   2 n.a. Table2 Consensus AA sequence of HC CDR2   3 n.a. Table 2 Consensus AAsequence of HC CDR3   4 n.a. Table 2 Consensus AA sequence of LC CDR1170 n.a. Table 2 Consensus AA sequence of LC CDR1   5 n.a. Table 2Consensus AA sequence of LC CDR2   6 n.a. Table 2 Consensus AA sequenceof LC CDR3   7 n.a. Table 2 Consensus AA sequence of HC FR1   8 n.a.Table 2 Consensus AA sequence of HC FR2   9 n.a. Table 2 Consensus AAsequence of HC FR3  10 n.a. Table 2 Consensus AA sequence of HC FR4  11n.a. Table 2 Consensus AA sequence of LC FR1  12 n.a. Table 2 ConsensusAA sequence of LC FR2  13 n.a. Table 2 Consensus AA sequence of LC FR3 14 n.a. Table 2 Consensus AA sequence of LC FR4  15 15 Table 3, AAsequence of HC VR of Ab VH15 Table A18; entire HC domain is in 4A, FIG.1; ID version is in FIG. 13; FIG. 2 NT sequence is in Example 1  28 28Table 3, AA sequence of LC VR of Ab VL28 Table A18; entire LC domain isin 4A FIG. 1; ID version is in FIG. 14; FIG. 3 NT sequence is in Example1  16 16 Table 3 AA sequence of HC VR of VH16 Table Abs 014, 028; IDversion is in 4A FIG. 13; NT sequence is in FIG. 2 Example 1  29 29Table 3 AA sequence of LC VR of Abs VL29 Table 014, 154, 157; ID versionis in 4A FIG. 14; NT sequence is in FIG. 3 Example 1  30 30 Table 3 AAsequence of LC VR of Abs VL30 Table 028, 155; ID version is in FIG. 4A14; NT sequence is in Example 1 FIG. 3  17 17 Table 3 AA sequence of HCVR of VH17 Table Abs 001, 009, 017, 025, 160, 4A 186, 187, 188, 189,190, 191, FIG. 2 192, 193, 202, 211; ID version is in FIG. 13;  31 31Table 3 AA sequence of LC VR of Abs VL31 Table 001, 002, 003; ID versionis in 4A FIG. 14; FIG. 3  18 18 Table 3 AA sequence of HC VR of VH18Table Abs 002, 010, B18, 026, 203, 4A 212; ID version is in FIG. 13;FIG. 2  19 19 Table 3 AA sequence of HC VR of VH19 Table Abs 003, 011,019, 027, 194, 4A 195, 196, 197, 198, 199, 200, FIG. 2 204, 213; IDversion is in FIG. 13;  32 32 Table 3 AA sequence of LC VR of Abs VL32Table 009, 010, 011; ID version is in 4A FIG. 14; FIG. 3  33 33 Table 3AA sequence of LC VR of Abs VL33 Table 017, B18, 019; ID version is in4A FIG. 14; FIG. 3  34 34 Table 3 AA sequence of LC VR of Abs VL34 Table025, 026, 027, 086; ID version 4A is in FIG. 14; FIG. 3  20 20 Table 3AA sequence of HC VR of Ab VH20 Table 086; ID version is in FIG. 13; 4AFIG. 2  21 21 Table 3 AA sequence of HC VR of VH21 Table Abs 154, 155;ID version is in 4A FIG. 13; FIG. 2  22 22 Table 3 AA sequence of HC VRof VH22 Table Abs 157, 159; ID version is in 4A FIG. 13; FIG. 2  35 35Table 3 AA sequence of LC VR of Ab VL35 Table 159; ID version is in FIG.14; 4A FIG. 3  36 36 Table 3 AA sequence of LC VR of Ab VL36 Table 160;ID version is in FIG. 14; 4A FIG. 3  37 37 Table 3 AA sequence of LC VRof Abs VL37 Table 186, 194; ID version is in FIG. 4A 14;  38 38 Table 3AA sequence of LC VR of Abs VL38 Table 187, 195; ID version is in FIG.4A 14; FIG. 3  39 39 Table 3 AA sequence of LC VR of Abs VL39 Table 188,196; ID version is in FIG. 4A 14; FIG. 3  40 40 Table 3 AA sequence ofLC VR of Abs VL40 Table 189, 197; ID version is in FIG. 4A 14; FIG. 3 41 41 Table 3 AA sequence of LC VR of Abs VL41 Table 190, 198; IDversion is in FIG. 4A 14; FIG. 3  42 42 Table 3 AA sequence of LC VR ofAbs VL42 Table 191, 199; ID version is in FIG. 4A 14;  43 43 Table 3 AAsequence of LC VR of Abs VL43 Table 192, 200; ID version is in FIG. 4A14; FIG. 3  44 44 Table 3 AA sequence of LC VR of Abs VL44 Table 193; IDversion is in FIG. 14; 4A FIG. 3  45 45 Table 3 AA sequence of LC VR ofAbs VL45 Table 202, 203, 204, 210, 031, 032, 4A 033, 034; ID version isin FIG. FIG. 3 14; NT sequence is in Example 1  46 46 Table 3 AAsequence of LC VR of Abs VL46 Table 211, 212, 213, 219, 037, 038, 4A039, 040; ID version is in FIG. FIG. 3 14;  23 23 Table 3 AA sequence ofHC VR of VH23 Table Abs 210, 219 ; ID version is in 4A FIG. 13; FIG. 2 24 24 Table 3 AA sequence of HC VR of VH24 Table Abs A001, A002, A003,A010, 4A A011, 031, 037; ID version is FIG. 2 in FIG. 13; NT sequence isin Example 1  47 47 Table 3 AA sequence of LC VR of Abs VL47 Table A001,004, 007, 016; ID 4A version is in FIG. 14; FIG. 3  48 48 Table 3 AAsequence of LC VR of Abs VL48 Table 002, 005, 008, A017; ID 4A versionis in FIG. 14; FIG. 3  25 25 Table 3 AA sequence of HC VR of VH25 TableAbs 004, 005, 006, 012, 013, 4A 032, 038, 043, 044, 045, 046, FIG. 2047, 048, 049, 050, 051, 052, 067, 068, 069, 070, 073, 074, 075, 076,077; ID version is in FIG. 13; NT sequence is in Example 1  49 49 Table3 AA sequence of LC VR of Abs VL49 Table A003, 006, A009, C18; ID 4Aversion is in FIG. 14; FIG. 3  26 26 Table 3 AA sequence of HC VR ofVH26 Table Abs 007, 008, A009, A14, 015, 4A 033, 039; ID version is inFIG. FIG. 2 13;  50 50 Table 3 AA sequence of LC VR of Abs VL50 TableA010 012, A14, A019; ID 4A version is in FIG. 14; FIG. 3  51 51 Table 3AA sequence of LC VR of Ab VL51 Table A011, 013, 015; ID version is 4Ain FIG. 14; FIG. 3  27 27 Table 3 AA sequence of HC VR of VH27 Table Abs016, A017, C18, A019, 4A 034, 040; ID version is in FIG. FIG. 2 13;  6060 Table 3 AA sequence of LC VR of Ab VL60 Table 043; ID version is inFIG. 14; 4A FIG. 3  52 52 Table 3 AA sequence of LC VR of Abs VL52 Table044, 071, 072, 078; ID version 4A is in FIG. 14; NT sequence is in FIG.3 Example 1  57 57 Table 3 AA sequence of LC VR of Ab VL57 Table 045; IDversion is in FIG. 14; 4A FIG. 3  59 59 Table 3 AA sequence of LC VR ofAb VL59 Table 046; ID version is in FIG. 14; 4A FIG. 3  55 55 Table 3 AAsequence of LC VR of Ab VL55 Table 047; ID version is in FIG. 14; 4AFIG. 3  58 58 Table 3 AA sequence of LC VR of Ab VL58 Table 048; IDversion is in FIG. 14; 4A FIG. 3  54 54 Table 3 AA sequence of LC VR ofAb VL54 Table 049; ID version is in FIG. 14; 4A FIG. 3  56 56 Table 3 AAsequence of LC VR of Ab VL56 Table 050; ID version is in FIG. 14; 4A  5353 Table 3 AA sequence of LC VR of Ab VL53 Table 051; ID version is inFIG. 14; 4A FIG. 3  61 61 Table 3 AA sequence of LC VR of Ab VL61 Table052; ID version is in FIG. 14; 4A FIG. 3 153 153  Table 3 AA sequence ofLC VR of Ab VL153 Table 067; ID version is in FIG. 14; 4A FIG. 3 154154  Table 3 AA sequence of LC VR of Ab VL154 Table 068; ID version isin FIG. 14; 4A FIG. 3 155 155  Table 3 AA sequence of LC VR of Abs VL155Table 069, 079; ID version is in FIG. 4A 14; 156 156  Table 3 AAsequence of LC VR of Ab VL156 Table 070; ID version is in FIG. 14; 4AFIG. 3 162 162  Table 3 AA sequence of HC VR of Ab VL162 Table 071 4AFIG. 17 163 163  Table 3 AA sequence of HC VR of Ab VL163 Table 072 4AFIG. 17 165 165  Table 3 AA sequence of LC VR of Ab VL165 Table 073 4AFIG. 17 166 166  Table 3 AA sequence of LC VR of Abs VL166 Table 074,080 4A FIG. 17 167 167  Table 3 AA sequence of LC VR of Ab VL167 Table075 4A FIG. 17 168 168  Table 3 AA sequence of LC VR of Ab VL168 Table076 4A FIG. 17 169 169  Table 3 AA sequence of LC VR of Abs VL169 Table077, 081 4A FIG. 17 164 164  Table 3 AA sequence of HC VR of VL164 TableAbs 078, 079, 080, 081 4A FIG. 17 161 HC161 Table AA sequence of HC VR4A consensus; ID version is in FIG. 2 FIG. 13;  62 LC62 Table AAsequence of LC VR 4A consensus; ID version is in FIG. 3 FIG. 14;  9615-ID Table AA sequence of HC VR of Ab 4B A18; non-ID version is in FIG.FIG. 13 2; 110 28-ID Table AA sequence of LC VR of Ab 4B A18; non-IDversion is in FIG. 3 FIG. 14  97 16-ID Table AA sequence of HC VR of 4BAbs 014, 028; non-ID version FIG. 13 is in FIG. 2; VSS 111 29-ID TableAA sequence of LC VR of Abs 4B 014, 154, 157; non-ID version FIG. 14 isin FIG. 3;  98 17-ID Table AA sequence of HC VR of Ab 4B 001, 009, 017,025, 160, 186, FIG. 13 187, 188, 189, 190, 191, 192, 193, 202, 211;non-ID version is in FIG. 2; 112 30-ID Table AA sequence of LC VR of Abs4B 028, 155; non-ID version is in FIG. 14 FIG. 3;  99 18-ID Table AAsequence of HC VR of 4B Abs 002, 010, B18, 026, 203, FIG. 13 212; non-IDversion is in FIG. 2; SS 113 35-ID Table AA sequence of LC VR of Ab 4B159; non-ID version is in FIG. FIG. 14 3; 100 19-ID Table AA sequence ofHC VR of 4B Abs 003, 011, 019, 027, 194, FIG. 13 195, 196, 197, 198,199, 200, 204, 213; non-ID version is in FIG. 2; 114 31-ID Table AAsequence of LC VR of Abs 4B 001, 002, 003; non-ID version FIG. 14 is inFIG. 3; 101 21-ID Table AA sequence of HC VR of 4B Abs 154, 155; non-IDversion FIG. 13 is in FIG. 2; 115 32-ID Table AA sequence of LC VR ofAbs 4B 009, 010, 011; non-ID version FIG. 14 is in FIG. 3; 102 22-IDTable AA sequence of HC VR of 4B Abs 157, 159; non-ID version FIG. 13 isin FIG. 2; 116 33-ID Table AA sequence of LC VR of Abs 4B 017, B18, 019;non-ID version FIG. 14 is in FIG. 3; 103 20-ID Table AA sequence of HCVR of Ab 4B 086; non-ID version is in FIG. FIG. 13 2; 117 34-ID Table AAsequence of LC VR of Abs 4B 025, 026, 027, 086; non-ID FIG. 14 versionis in FIG. 3; 104 23-ID Table AA sequence of HC VR of 4B Abs 210, 219;non-ID version FIG. 13 is in FIG. 2; 118 36-ID Table AA sequence of LCVR of Ab 4B 160; non-ID version is in FIG. FIG. 14 3; 105 24-ID Table AAsequence of HC VR of 4B Abs A001, A002, A003, A010, FIG. 13 A011, 031,037; non-ID version is in FIG. 2; 119 45-ID Table AA sequence of LC VRof Abs 4B 202, 203, 204, 210, 031, 032, FIG. 14 033, 034; non-ID versionis in FIG. 3; 106 25-ID Table AA sequence of HC VR of 4B Abs 004, 005,006, 012, 013, FIG. 13 032, 038, 043, 044, 045, 046, 047, 048, 049, 050,051, 052, 067, 068, 069, 070, 073, 074, 075, 076, 077; non-ID version isin FIG. 2; 120 46-ID Table AA sequence of LC VR of Abs 4B 211, 212, 213,219, 037, 038, FIG. 14 039, 040; non-ID version is in FIG. 3; 107 26-IDTable AA sequence of HC VR of 4B Abs 007, 008, A009, A14, 015, FIG. 13033, 039; non-ID version is in FIG. 2; 121 37-ID Table AA sequence of LCVR of Abs 4B 186, 194; non-ID version is in FIG. 14 FIG. 3; 108 27-IDTable AA sequence of HC VR of 4B Abs 016, A017, C18, A019, FIG. 13 034,040; non-ID version is in FIG. 2; 122 38-ID Table AA sequence of LC VRof Abs 4B 187, 195; non-ID version is in FIG. 14 FIG. 3; 109 161-ID Table AA sequence of HC VR 4B consensus ID; non-ID version FIG. 13 is inFIG. 2; 123 39-ID Table AA sequence of LC VR of Abs 4B 188, 196; non-IDversion is in FIG. 14 FIG. 3; 124 40-ID Table AA sequence of LC VR ofAbs 4B 189, 197; non-ID version is in FIG. 14 FIG. 3; 125 41-ID Table AAsequence of LC VR of Abs 4B 190, 198; non-ID version is in FIG. 14 FIG.3; 126 42-ID Table AA sequence of LC VR of Abs 4B 191, 199; non-IDversion is in FIG. 14 FIG. 3; 127 43-ID Table AA sequence of LC VR ofAbs 4B 192, 200; non-ID version is in FIG. 14 FIG. 3; 128 44-ID Table AAsequence of LC VR of Abs 4B 193; non-ID version is in FIG. FIG. 14 3;129 47-ID Table AA sequence of LC VR of Abs 4B A001, 004, 007, 016 FIG.14 130 48-ID Table AA sequence of LC VR of Abs 4B 002, 005, 008, A017;non-ID FIG. 14 version is in FIG. 3; 131 49-ID Table AA sequence of LCVR of Abs 4B A003, 006, A009, C18; non-ID FIG. 14 version is in FIG. 3;132 50-ID Table AA sequence of LC VR of Abs 4B A010 012, A14, A019;non-ID FIG. 14 version is in FIG. 3; 133 51-ID Table AA sequence of LCVR of Ab 4B A011, 013, 015; non-ID FIG. 14 version is in FIG. 3; 13452-ID Table AA sequence of LC VR of Abs 4B 044, 071, 072, 078; non-IDFIG. 14 version is in FIG. 3; 135 53-ID Table AA sequence of LC VR of Ab4B 051; non-ID version is in FIG. FIG. 14 3; 136 54-ID Table AA sequenceof LC VR of Ab 4B 049; non-ID version is in FIG. FIG. 14 3; 137 55-IDTable AA sequence of LC VR of Ab 4B 047; non-ID version is in FIG. FIG.14 3; 138 56-ID Table AA sequence of LC VR of Ab 4B 050; non-ID versionis in FIG. FIG. 14 3; 139 57-ID Table AA sequence of LC VR of Ab 4B 045;non-ID version is in FIG. FIG. 14 3; 140 58-ID Table AA sequence of LCVR of Ab 4B 048; non-ID version is in FIG. FIG. 14 3; 141 59-ID Table AAsequence of LC VR of Ab 4B 046; non-ID version is in FIG. FIG. 14 3; 14260-ID Table AA sequence of LC VR of Ab 4B 043; non-ID version is in FIG.FIG. 14 3; 143 61-ID Table AA sequence of LC VR of Ab 4B 052; non-IDversion is in FIG. FIG. 14 3; 157 153-ID  Table AA sequence of LC VR ofAb 4B 067; non-ID version is in FIG. FIG. 14 3; 158 154-ID  Table AAsequence of LC VR of Ab 4B 068; non-ID version is in FIG. FIG. 14 3; 159155-ID  Table AA sequence of LC VR of Abs 4B 069, 079; non-ID version isin FIG. 14 FIG. 3; 160 156-ID  Table AA sequence of LC VR of Ab 4B 070;non-ID version is in FIG. FIG. 14 3; 144 62-ID Table AA sequence of LCVR 4B consensus ID; non-ID version FIG. 14 is in FIG. 3;  63 VH16Example 1 NT sequence of HC VR of Abs 014, 028  64 VL29 Example 1 NTsequence of LC VR of Abs 014, 154, 157  65 VL30 Example 1 NT sequence ofLC VR of Abs 028, 155  66 VH15 Example 1 NT sequence of HC VR of Ab A18187 VL28 Example 1 NT sequence of LC VR of Ab A18 149 VL52 Example 1 NTsequence of LC VR of Abs 044, 071, 072, 078 150 VL45 Example 1 NTsequence of LC VR of Abs 202, 203, 204, 210, 031, 032, 033, 034 151 VH25Example 1 NT sequence of HC VR of Abs 004, 005, 006, 012, 013, 032, 038,043, 044, 045, 046, 047, 048, 049, 050, 051, 052, 067, 068, 069, 070,073, 074, 075, 076, 077 152 VH24 Example 1 NT sequence of HC VR of AbsA001, A002, A003, A010, A011, 031, 037  94 15 FIG. 1 AA sequence of HCof Ab A18 188 28 FIG. 1 AA sequence of LC of Ab A18 145 n.a. see text AAsequence of LC CDR1 of Ab 146 n.a. see text AA sequence of LC CDR1 ofFI6 VK 147 n.a. see text AA sequence of LC CDR2 of FI6 VK 148 n.a. seetext AA sequence of LC CDR3 of FI6 VK  68 n.a. see text AA sequence ofHC CDR1 of Abs 044, 069, 032, 031  69 n.a. see text AA sequence of HCCDR2 of Abs 044, 069, 032, 031  70 n.a. see text AA sequence of HC CDR3of Abs 044, 069, 032, 031  71 n.a. see text AA sequence of LC CDR1 ofAbs 032, 031  72 n.a. see text AA sequence of LC CDR2 of Abs 044, 069,032, 031  73 n.a. see text AA sequence of LC CDR3 of Abs 044, 069, 032,031  74 n.a. see text AA sequence of HC FR1 of Ab 069  75 n.a. see textAA sequence of HC FR2 of Ab 069  76 n.a. see text AA sequence of HC FR3of Ab 069  77 n.a. see text AA sequence of HC FR4 of Ab 069  78 n.a. seetext AA sequence of LC FR1 of Ab 069  79 n.a. see text AA sequence of LCFR2 of Ab 069  80 n.a. see text AA sequence of LC FR3 of Ab 069  81 n.a.see text AA sequence of LC FR4 of Ab 069  82 n.a. see text AA sequenceof HC FR1 of Ab 031  83 n.a. see text AA sequence of LC CDR1 of Ab A18et al.  84 n.a. see text AA sequence of LC CDR2 of Ab A18 et al.  85n.a. see text AA sequence of LC CDR3 of Ab A18 et al.  86 n.a. see textAA sequence of HC CDR1 of Ab A18 et al.  87 n.a. see text AA sequence ofHC CDR2 of Ab A18 et al.  88 n.a. see text AA sequence of an HC CDR3  89n.a. see text AA sequence of HC FR4 of Ab A18 et al.  90 n.a. see textAA sequence of LC FR1 of Ab A18 et al.  91 n.a. see text AA sequence ofLC FR2 of Ab A18 et al.  92 n.a. see text AA sequence of LC FR3 of AbA18 et al.  93 n.a. see text AA sequence of LC FR4 of Ab A18 et al. 171n.a. see text AA sequence of HC FR4 of Ab 078 et al 172 n.a. see text AAsequence of LC CDR1 of Ab 069 173 n.a. see text AA sequence of H3 HA1174 n.a. see text AA sequence of H3 HA2 175 n.a. FIG. 12 AA sequence ofHC VR of FI6 176 n.a. FIG. 12 AA sequence of HC VR of FI370 177 n.a.FIG. 12 AA sequence of HC VR of FI6 variant 1 178 n.a. FIG. 12 AAsequence of HC VR of FI6 variant 3 179 n.a. FIG. 12 AA sequence of HC VRof FI6/370 180 n.a. FIG. 12 AA sequence of kappa LC VR of FI6 181 Seetext AA sequence of H1 HA1 182 See text AA sequence of H1 HA2 SEQ ID NO.Sequence 1 [S/T]Y[A/G]MH 2 V[I/V/L]S[Y/F]DG[S/N][Y/N][K/R]YYADSVQG 3D[S/T][R/K/Q]LR[S/T]LLYFEWLS[Q/S]G[Y/L/V][F/L][N/D][P/Y] 4Q[S/T][V/L/I][T/S][Y/F/W][N/S/D]YKNYLA 170Q[S/T][V/L/I][T/S][Y/F/W][N/S/D/Q/R/E]YKNYLA 5W[A/G]S[T/A/Y/H/K/D][R/L]E[S/T] 6 QQ[Y/H]YRTPP[T/S] 7[E/Q]VQLLE[S/T]GGGLVKPGQSLKLSCAASGFTF[S/T] 8 WVRQPPGKGLEWVA 9RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 10 WG[A/Q]G[T/A][T/M][L/V]TVSS 11[E/D]I[V/Q]MTQSP[D/S][S/T][L/V][A/S][V/A][S/T][L/V/R]G[E/D]R [A/V][T/S]I[N/T/Q/D/R/]C[K/R]SS 12 WYQQKPG[Q/K][P/A]PKLLIY 13GVP[D/E/S]RFSGSGSGTDFTLTISSLQ[A/P]ED[V/F/K/D]A[V/T]YYC 14FG[G/Q/T/S/N]GTK[L/V][D/E]IK 15EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSS 28EIVMTQSPDSLAVSLGERATINCKSSQSVTYNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQYYRTPPTFGGGTKLDIK 16EVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTKLRSLLYFEWLSSGLLDYWGQGAMVTVSS 29EIVMTQSPDSLAVSLGERATINCKSSQSVTFSYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQYYRTPPTFGGGTKLDIK 30EIVMTQSPDSLAVSLGERATINCKSSQSVTFDYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQYYRTPPTFGGGTKLDIK 17EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSS 31EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGGGTKLDIK 18EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVLSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSS 19EVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVLSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSS 32EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGGGTKLDIK 33EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQHYRTPPSFGGGTKLDIK 34EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQHYRTPPSFGGGTKLDIK 20EVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVVSFDGNNRYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRSLLYFEWLSSGVLDYWGQGAMVTVSS 21EVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGNNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVTV SS 22EVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVVSYDGNNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVTV SS 35EIVMTQSPDSLAVSLGERATINCKSSQSVTWSYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQYYRTPPTFGGGTKLDIK 36EIVMSQSPDTLAVTLGERASINCKSSQTVTFNYKNYLAWYQQKPGQPPKVLIYWASARETGVPERFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGQGTKLEIK 37EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGTGTKLDIK 38EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGSGTKLDIK 39EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGQGTKLDIK 40EIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGNGTKLDIK 41EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGTGTKLDIK 42EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGSGTKLDIK 43EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGQGTKLDIK 44EIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQHYRTPPSFGNGTKLDIK 45DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 46DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLGWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 23EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYPEWLSQGYFNPWGAGTTLTVSS 24EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLTVSS 47DIVMTQSPDTLAVTLGERATIQCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTITSLQAEDVA VYYCQQHYRTPPSFGQGTKLDIK 48DIVMTQSPDTVAVTVGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPSFGQGTKLDIK 25QVQLLETGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLTVSS 49DIVMTQSPDTVAVTLGERATIDCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPSFGQGTKLDIK 26EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRTLLYFEWLSQGYFNPWGQGTTLTVSS 50DIVMTQSPDTLAVTVGERATIRCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPSFGQGTKLDIK 51DIVMTQSPDTLAVSRGERATIDCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDEA VYYCQQHYRTPPSFGQGTKLDIK 27EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRTLLYPEWLSQGYFDPWGQGTTLTVSS 60DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQYYRTPPSFGQGTKVEIK 52DIQMTQSPSSLSASVGDRVTITCRSSQSITFDYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 57DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQQHYRTPPSFGQGTKVEIK 59DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDDATY YCQQHYRTPPSFGQGTKVEIK 55DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSKLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 58DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDKATY YCQQHYRTPPSFGQGTKVEIK 54DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSHLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 56DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSDLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 53DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 61DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 153DIQMTQSPSSLSASVGDRVTITCRSSQSITFQYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 154DIQMTQSPSSLSASVGDRVTITCRSSQSITFRYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 155DIQMTQSPSSLSASVGDRVTITCRSSQSITFEYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 156DIQMTQSPSSLSASVGDRVTITCRSSQSITFDYKNYLAWYQQKPGKAPKLLIYWGSTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 162EVQLLESGGGLVKPGQSLKLSCAASGFSFSTYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADTVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLTVSS 163EVQLLESGGGLRKPGQSLKLSCAASGFSFSTYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLTVSS 165DIQMTQSPSSLSASVGDRVTITCRSSQSITWNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQHYRTPPSFGQGTKVEIK 166DIQMTQSPSSLSASVGDRVTITCRSSQSITWDYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQHYRTPPSFGQGTKVEIK 167DIQMTQSPSSLSASVGDRVTITCRSSQSITWQYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQHYRTPPSFGQGTKVEIK 168DIQMTQSPSSLSASVGDRVTITCRSSQSITWRYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQHYRTPPSFGQGTKVEIK 169DIQMTQSPSSLSASVGDRVTITCRSSQSITWEYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQHYRTPPSFGQGTKVEIK 164QVQLLETGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTVTVSS 161EVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVTVSS 62DIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYRTPPSFGQGTKVEIK 96IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSS 110IDEIVMTQSPDSLAVSLGERATINCKSSQSVTYNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYRTPPTFGGGTKLDIK97 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDTKLRSLLYFEWLSSGLLDYWGQGAMVT 111IDEIVMTQSPDSLAVSLGERATINCKSSQSVTFSYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYRTPPTFGGGTKLDIK98 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTV SS 112IDEIVMTQSPDSLAVSLGERATINCKSSQSVTFDYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYRTPPTFGGGTKLDIK99 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVLSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTV 113IDEIVMTQSPDSLAVSLGERATINCKSSQSVTWSYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYRTPPTFGGGTKLDIK100 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVLSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTV SS 114IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGGGTKLDIK101 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGNNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVT VSS 115IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGGGTKLDIK102 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVVSYDGNNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVT VSS 116IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPSFGGGTKLDIK103 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTTYAMHWVRQPPGKGLEWVAVVSFDGNNRYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRSLLYPEWLSSGVLDYWGQGAMVTV SS 117IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPSFGGGTKLDIK104 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSQGYFNPWGAGTTLTV SS 118IDEIVMSQSPDTLAVTLGERASINCKSSQTVTFNYKNYLAWYQQKPGQPPKVLIYWASARETGVPERFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGQGTKLEIK105 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLTV SS 119IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK106 IDQVQLLETGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGQGTTLT VSS 120IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLGWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK107 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRTLLYFEWLSQGYFNPWGQGTTLTV SS 121IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGTGTKLDIK108 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFTSYAMHWVRQPPGKGLEWVAVVSYDGNYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRTLLYFEWLSQGYFDPWGQGTTLTV SS 122IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGSGTKLDIK109 IDEVQLLESGGGLVKPGQSLKLSCAASGFTFSSYGMHWVRQPPGKGLEWVAVVSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSKLRSLLYFEWLSSGLLDYWGQGAMVT VSS 123IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGQGTKLDIK124 IDEIVMTQSPDSLAVSLGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGNGTKLDIK125 IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGTGTKLDIK126 IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGSGTKLDIK127 IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGQGTKLDIK128 IDEIVMTQSPDSLAVSLGERATINCKSSQTLSFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGNGTKLDIK129 IDDIVMTQSPDTLAVTLGERATIQCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTITSLQAEDV AVYYCQQHYRTPPSFGQGTKLDIK130 IDDIVMTQSPDTVAVTVGERATINCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQHYRTPPSFGQGTKLDIK131 IDDIVMTQSPDTVAVTLGERATIDCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQHYRTPPSFGQGTKLDIK132 IDDIVMTQSPDTLAVTVGERATIRCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDE AVYYCQQHYRTPPSFGQGTKLDIK133 IDDIVMTQSPDTLAVSRGERATIDCKSSQTVTFNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDE AVYYCQQHYRTPPSFGQGTKLDIK134 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFDYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK135 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSTLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK136 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSHLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK137 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSKLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK138 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSDLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK139 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDVAT YYCQQHYRTPPSFGQGTKVEIK140 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDKAT YYCQQHYRTPPSFGQGTKVEIK141 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDDAT YYCQQHYRTPPSFGQGTKVEIK142 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYYRTPPSFGQGTKVEIK143 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK157 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFQYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK158 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFRYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK159 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFEYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK160 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFDYKNYLAWYQQKPGKAPKLLIYWGSTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK144 IDDIQMTQSPSSLSASVGDRVTITCRSSQSITFNYKNYLAWYQQKPGKAPKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQHYRTPPSFGQGTKVEIK 63GAGGTACAGCTCCTCGAATCGGGAGGGGGACTGGTCAAACCCGGTCAATCGCTCAAACTCTCGTGTGCAGCGTCAGGTTTTACGTTCAGCTCATATGGGATGCACTGGGTCCGCCAGCCTCCGGGAAAGGGACTGGAGTGGGTGGCAGTCGTGTCGTATGACGGGAGCAATAAGTACTACGCCGATTCAGTGCAAGGTCGGTTTACCATTTCGAGGGATAACAGCAAGAACACGCTCTACTTGCAGATGAACTCACTTAGAGCGGAAGATACGGCTGTGTACTATTGCGCCAAAGACACAAAGCTGCGATCCCTGTTGTACTTCGAATGGTTGTCCTCGGGCTTGCTTGACTATTGGGGGCAGGGCGCCATGGTCACAG TATCCAGCGCGTCGACTAAGGGGCCC 64GAGATCGTGATGACGCAGAGCCCCGATAGCCTCGCTGTCTCATTGGGGGAACGGGCCACGATTAACTGCAAATCCTCACAGTCGGTGACTTTCAGCTATAAGAATTACCTGGCATGGTATCAGCAGAAGCCGGGTCAACCCCCAAAACTGTTGATCTACTGGGCCTCCACACGCGAGTCGGGAGTCCCGGACCGATTTTCGGGTTCAGGGTCCGGCACTGACTTTACCCTCACAATTTCATCGCTTCAAGCGGAGGATGTAGCAGTGTACTATTGTCAGCAGTATTACAGAACACCTCCCACCTTCGGAGGGGGAACGAAACTTGACATCAAGGGAT CC 65 NT:GAGATCGTGATGACGCAGAGCCCCGATAGCCTCGCTGTCTCATTGGGGGAACGGGCCACGATTAACTGCAAATCCTCACAGTCGGTGACTTTCGACTATAAGAATTACCTGGCATGGTATCAGCAGAAGCCGGGTCAACCCCCAAAACTGTTGATCTACTGGGCCTCCACACGCGAGTCGGGAGTCCCGGACCGATTTTCGGGTTCAGGGTCCGGCACTGACTTTACCCTCACAATTTCATCGCTTCAAGCGGAGGATGTAGCAGTGTACTATTGTCAGCAGTATTACAGAACACCTCCCACCTTCGGAGGGGGAACGAAACTTGACATCAAGGGAT CC 66GAAGTGCAACTCCTCGAGTCAGGAGGAGGTTTGGTGAAACCGGGTCAGTCCTTGAAACTGAGCTGTGCAGCAAGCGGGTTCACGTTTACGTCGTACGGCATGCACTGGGTACGGCAGCCTCCCGGGAAGGGACTTGAATGGGTCGCCGTCATCTCATACGACGGGTCGTACAAATACTATGCGGATAGCGTGCAAGGTCGCTTCACAATTTCCCGGGACAATTCGAAGAATACACTGTATCTTCAGATGAACTCGCTCAGGGCTGAGGACACGGCGGTCTATTACTGCGCGAAGGATTCGCGACTCAGATCCCTTTTGTACTTTGAGTGGCTGTCGCAGGGGTATTTCAACCCATGGGGAGCCGGAACCACTTTGACCG TATCAAGCGCGTCAACAAAGGGGCCC187 GAAATTGTAATGACGCAGAGCCCTGATAGCCTTGCCGTGTCCCTGGGTGAGAGGGCGACAATCAATTGTAAGTCATCACAGTCGGTCACGTACAACTACAAGAACTACCTGGCGTGGTATCAACAGAAACCCGGGCAGCCGCCCAAATTGCTCATCTATTGGGCTTCGACACGGGAGTCGGGTGTGCCAGACCGCTTCTCCGGGTCAGGATCGGGAACTGACTTCACGTTGACTATTTCGTCCCTCCAGGCAGAAGATGTAGCCGTCTACTATTGCCAACAGTATTACAGAACGCCGCCTACATTTGGAGGCGGGACCAAACTTGACATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGA CCAAGAGCTTCAACCGGGGCGAGTG 149GACATTCAGATGACTCAGTCGCCTTCGTCATTGTCCGCCTCCGTGGGTGATAGGGTCACGATCACGTGCCGGAGCAGCCAGTCCATCACCTTCAATTACAAAAACTATTTGGCATGGTATCAACAGAAACCCGGAAAGGCGCCGAAGCTCCTGATCTACTGGGGTTCATATCTTGAGTCGGGGGTGCCGTCGAGATTTTCGGGCAGCGGATCAGGGACGGATTTCACGCTGACCATTTCGTCACTCCAGCCCGAGGACTTTGCGACATATTACTGTCAACAGCACTACAGGACACCCCCATCTTTCGGACAGGGGACTAAAGTAGAAATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGA CCAAGAGCTTCAACCGGGGCGAGTGCTGA150 GACATTCAGATGACTCAGTCGCCTTCGTCATTGTCCGCCTCCGTGGGTGATAGGGTCACGATCACGTGCCGGAGCAGCCAGTCCATCACCTTCAATTACAAAAACTATTTGGCATGGTATCAACAGAAACCCGGAAAGGCGCCGAAGCTCCTGATCTACTGGGGTTCATATCTTGAGTCGGGGGTGCCGTCGAGATTTTCGGGCAGCGGATCAGGGACGGATTTCACGCTGACCATTTCGTCACTCCAGCCCGAGGACTTTGCGACATATTACTGTCAACAGCACTACAGGACACCCCCATCTTTCGGACAGGGGACTAAAGTAGAAATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGAGAATTC 151CAGGTACAATTGCTTGAGACAGGTGGAGGACTCGTGAAGCCAGGTCAGTCATTGAAACTGAGCTGTGCCGCATCCGGGTTCACATTCACTTCCTACGCGATGCACTGGGTCCGCCAGCCTCCCGGAAAGGGACTTGAGTGGGTCGCTGTGGTATCGTATGATGGGAATTACAAATACTATGCAGACTCCGTGCAAGGCCGGTTTACGATTAGCAGGGACAACTCGAAGAATACCCTTTACCTCCAAATGAACTCGCTCCGAGCGGAGGACACGGCGGTGTATTACTGCGCGAAGGATTCACGGTTGAGATCGCTGCTCTATTTTGAATGGTTGTCACAGGGGTACTTCAACCCGTGGGGTCAGGGAACAACACTGACCGTCAGCTCAGCCTCGACTAAAGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGGTACTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 152GAAGTACAATTGCTTGAGTCGGGTGGAGGACTCGTGAAGCCAGGTCAGTCATTGAAACTGAGCTGTGCCGCATCCGGGTTCACATTCACTTCCTACGCGATGCACTGGGTCCGCCAGCCTCCCGGAAAGGGACTTGAGTGGGTCGCTGTGGTATCGTATGATGGGAATTACAAATACTATGCAGACTCCGTGCAAGGCCGGTTTACGATTAGCAGGGACAACTCGAAGAATACCCTTTACCTCCAAATGAACTCGCTCCGAGCGGAGGACACGGCGGTGTATTACTGCGCGAAGGATTCACGGTTGAGATCGCTGCTCTATTTTGAATGGTTGTCACAGGGGTACTTCAACCCGTGGGGTCAGGGAACAACACTGACCGTCAGCTCAGCCTCGACTAAAGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACGGTACCCACACATGCCCACCGTGCCCAGGTACTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 94EVQLLESGGGLVKPGQSLKLSCAASGFTFTSYGMHWVRQPPGKGLEWVAVISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSRLRSLLYFEWLSQGYFNPWGAGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPGTELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGEPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 188EIVMTQSPDSLAVSLGERATINCKSSQSVTYNYKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYRTPPTFGGGTKLDIKGSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE 145 QSITFDYKNYLA 044 146KSSQSVTFNYKNYLA 147 WASARES 148 QQHYRTPPT 68 SYAMH 69 VVSYDGNYKYYADSVQG70 DSRLRSLLYFEWLSQGYFNP 71 QSITFNYKNYLA 72 WGSYLES 73 QQHYRTPPS 74QVQLLETGGGLVKPGQSLKLSCAASGFTFT 75 WVRQPPGKGLEWVA 76RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 77 WGQGTTLTVSS 78DIQMTQSPSSLSASVGDRVTITCRSS 79 WYQQKPGKAPKLLIY 80GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 81 FGQGTKVEIK 82EVQLLESGGGLVKPGQSLKLSCAASGFTFT 83 KSSQSVTYNYKNYLA 84 WASTRES 85QQYYRTPPT 86 SYGMH 87 VISYDGSYKYYADSVQG 88 DSELRSLLYFEWLSQGYFNP 89WGAGTTLTVSS 90 EIVMTQSPDSLAVSLGERATINC 91 WYQQKPGQPPKLLIY 92GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 93 FGGGTKLDIK 171 WGQGTTVTVSS 172QSITFEYKNYLA 173 QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQN TLKLATGMRNVPEKQTR 174GLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEEMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKG 175QVQLVQSGGGVVQPGRSLRLSCVASGFTFSTYAMHWVRQAPGRGLEWVAVISYDGNYKYYADSVKGRFSISRDNSNNTLHLEMNTLRTEDTALYYCAKDSQLRSLLYFEWLSQGYFDPWGQGTLVTVTS 176QVQLVQSGGGVVPPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVISYDGNYKYYADSVRGRFTISRDNSKNTLNLDMNSLRTEDTALYYCAKDSQLRSLLYFDWLSQGYFDHWGQGTLVTVSS 177QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRSLLYFDWLSQGYFDYWGQGTLVTVSS 178QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKGLEWVAVISYDANYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSQLRSLLYFEWLSQGYFDYWGQGTLVTVSS 179QVQLVQSGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVISYDGNYKYYADSVKGRFTISRDNSKNTLYLEMNSLRTEDTALYYCAKDSQLRSLLYFDWLSQGYFDHWGQGTLVTVSS 180DIQMTSQPDSLAVSLGARATINCKSSQSVTFNYKNYLAWYQQKPGQPPKVLIYWASARESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQHYRTPPTFGQGTKVEIK181 TNADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHPVTIGECPKYV RSTKLRMATGLRNIPSIQS 182GLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDACMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQ CRICI

EXAMPLES Example 1. Designing of Anti-HA Antibodies

Human antibodies (IgG) targeting viral hemagglutinin (HA) werecomputationally designed. HA mediates viral binding to host cell surfacereceptor, and cell membrane fusion to the viral envelope, resulting inviral entry. The antibody molecules described herein were designed toblock HA's fusogenic activity.

All antibody constructs were based on human IgG1 structure (γ1 heavychain and κ light chain). Point mutations in the V_(H) (variable heavydomain) and V_(L) (variable light domain) were computationally designed.These mutations are located within or outside the CDRs (ComplementarityDetermining Regions). The mutations were designed, e.g., to modifyantigen binding properties (e.g., for stronger or weaker bindingaffinity), or to stabilize structure, or to improve expressionproperties, etc.

The heavy and light chain sequences of one antibody, called A18 isprovided in FIG. 1.

The heavy and light chain pairings for exemplary computationallydesigned antibodies are shown in Table 3, above in Detailed Description.

DNA sequences for the variable heavy chain and variable light chain foreach of antibodies Ab A18, Ab 031, Ab 032, Ab 044, Ab 014 and Ab 028 areprovided below.

VH16: (SEQ ID NO: 63)GAGGTACAGCTCCTCGAATCGGGAGGGGGACTGGTCAAACCCGGTCAATCGCTCAAACTCTCGTGTGCAGCGTCAGGTTTTACGTTCAGCTCATATGGGATGCACTGGGTCCGCCAGCCTCCGGGAAAGGGACTGGAGTGGGTGGCAGTCGTGTCGTATGACGGGAGCAATAAGTACTACGCCGATTCAGTGCAAGGTCGGTTTACCATTTCGAGGGATAACAGCAAGAACACGCTCTACTTGCAGATGAACTCACTTAGAGCGGAAGATACGGCTGTGTACTATTGCGCCAAAGACACAAAGCTGCGATCCCTGTTGTACTTCGAATGGTTGTCCTCGGGCTTGCTTGACTATTGGGGGCAGGGCGCCATGGTCACAGTATCCAGCGCGTCGACTAAG GGGCCC VL29:(SEQ ID NO: 64)GAGATCGTGATGACGCAGAGCCCCGATAGCCTCGCTGTCTCATTGGGGGAACGGGCCACGATTAACTGCAAATCCTCACAGTCGGTGACTTTCAGCTATAAGAATTACCTGGCATGGTATCAGCAGAAGCCGGGTCAACCCCCAAAACTGTTGATCTACTGGGCCTCCACACGCGAGTCGGGAGTCCCGGACCGATTTTCGGGTTCAGGGTCCGGCACTGACTTTACCCTCACAATTTCATCGCTTCAAGCGGAGGATGTAGCAGTGTACTATTGTCAGCAGTATTACAGAACACCTCCCACCTTCGGAGGGGGAACGAAACTTGACATCAAGGGATCC VL30: (SEQ ID NO:65) GAGATCGTGATGACGCAGAGCCCCGATAGCCTCGCTGTCTCATTGGGGGAACGGGCCACGATTAACTGCAAATCCTCACAGTCGGTGACTTTCGACTATAAGAATTACCTGGCATGGTATCAGCAGAAGCCGGGTCAACCCCCAAAACTGTTGATCTACTGGGCCTCCACACGCGAGTCGGGAGTCCCGGACCGATTTTCGGGTTCAGGGTCCGGCACTGACTTTACCCTCACAATTTCATCGCTTCAAGCGGAGGATGTAGCAGTGTACTATTGTCAGCAGTATTACAGAACACCTCCCACCTTCGGAGGGGGAACGAAACTTGACATCAAGGGATCC VH15: (SEQ ID NO:66) GAAGTGCAACTCCTCGAGTCAGGAGGAGGTTTGGTGAAACCGGGTCAGTCCTTGAAACTGAGCTGTGCAGCAAGCGGGTTCACGTTTACGTCGTACGGCATGCACTGGGTACGGCAGCCTCCCGGGAAGGGACTTGAATGGGTCGCCGTCATCTCATACGACGGGTCGTACAAATACTATGCGGATAGCGTGCAAGGTCGCTTCACAATTTCCCGGGACAATTCGAAGAATACACTGTATCTTCAGATGAACTCGCTCAGGGCTGAGGACACGGCGGTCTATTACTGCGCGAAGGATTCGCGACTCAGATCCCTTTTGTACTTTGAGTGGCTGTCGCAGGGGTATTTCAACCCATGGGGAGCCGGAACCACTTTGACCGTATCAAGCGCGTCAACAAAG GGGCCC VL28:(SEQ ID NO: 67)GAAATTGTAATGACGCAGAGCCCTGATAGCCTTGCCGTGTCCCTGGGTGAGAGGGCGACAATCAATTGTAAGTCATCACAGTCGGTCACGTACAACTACAAGAACTACCTGGCGTGGTATCAACAGAAACCCGGGCAGCCGCCCAAATTGCTCATCTATTGGGCTTCGACACGGGAGTCGGGTGTGCCAGACCGCTTCTCCGGGTCAGGATCGGGAACTGACTTCACGTTGACTATTTCGTCCCTCCAGGCAGAAGATGTAGCCGTCTACTATTGCCAACAGTATTACAGAACGCCGCCTACATTTGGAGGCGGGACCAAACTTGACATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC VL52: (SEQ ID NO: 149)GACATTCAGATGACTCAGTCGCCTTCGTCATTGTCCGCCTCCGTGGGTGATAGGGTCACGATCACGTGCCGGAGCAGCCAGTCCATCACCTTCAATTACAAAAACTATTTGGCATGGTATCAACAGAAACCCGGAAAGGCGCCGAAGCTCCTGATCTACTGGGGTTCATATCTTGAGTCGGGGGTGCCGTCGAGATTTTCGGGCAGCGGATCAGGGACGGATTTCACGCTGACCATTTCGTCACTCCAGCCCGAGGACTTTGCGACATATTACTGTCAACAGCACTACAGGACACCCCCATCTTTCGGACAGGGGACTAAAGTAGAAATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGA VL45: (SEQ ID NO: 150)GACATTCAGATGACTCAGTCGCCTTCGTCATTGTCCGCCTCCGTGGGTGATAGGGTCACGATCACGTGCCGGAGCAGCCAGTCCATCACCTTCAATTACAAAAACTATTTGGCATGGTATCAACAGAAACCCGGAAAGGCGCCGAAGCTCCTGATCTACTGGGGTTCATATCTTGAGTCGGGGGTGCCGTCGAGATTTTCGGGCAGCGGATCAGGGACGGATTTCACGCTGACCATTTCGTCACTCCAGCCCGAGGACTTTGCGACATATTACTGTCAACAGCACTACAGGACACCCCCATCTTTCGGACAGGGGACTAAAGTAGAAATCAAGGGATCCGTGGCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGAGAATTC VH25: (SEQ ID NO: 151)CAGGTACAATTGCTTGAGACAGGTGGAGGACTCGTGAAGCCAGGTCAGTCATTGAAACTGAGCTGTGCCGCATCCGGGTTCACATTCACTTCCTACGCGATGCACTGGGTCCGCCAGCCTCCCGGAAAGGGACTTGAGTGGGTCGCTGTGGTATCGTATGATGGGAATTACAAATACTATGCAGACTCCGTGCAAGGCCGGTTTACGATTAGCAGGGACAACTCGAAGAATACCCTTTACCTCCAAATGAACTCGCTCCGAGCGGAGGACACGGCGGTGTATTACTGCGCGAAGGATTCACGGTTGAGATCGCTGCTCTATTTTGAATGGTTGTCACAGGGGTACTTCAACCCGTGGGGTCAGGGAACAACACTGACCGTCAGCTCAGCCTCGACTAAAGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGGTACTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA VH24: (SEQ ID NO: 152)GAAGTACAATTGCTTGAGTCGGGTGGAGGACTCGTGAAGCCAGGTCAGTCATTGAAACTGAGCTGTGCCGCATCCGGGTTCACATTCACTTCCTACGCGATGCACTGGGTCCGCCAGCCTCCCGGAAAGGGACTTGAGTGGGTCGCTGTGGTATCGTATGATGGGAATTACAAATACTATGCAGACTCCGTGCAAGGCCGGTTTACGATTAGCAGGGACAACTCGAAGAATACCCTTTACCTCCAAATGAACTCGCTCCGAGCGGAGGACACGGCGGTGTATTACTGCGCGAAGGATTCACGGTTGAGATCGCTGCTCTATTTTGAATGGTTGTCACAGGGGTACTTCAACCCGTGGGGTCAGGGAACAACACTGACCGTCAGCTCAGCCTCGACTAAAGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACGGTACCCACACATGCCCACCGTGCCCAGGTACTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Each of the above sequences can be modified to include an ATCGATnucleotide sequence at the 5′ end, which will encode a variable heavychain or light chain polypeptide comprising Ile-Asp at the aminoterminus.

Example 2. Binding of Anti-HA Antibodies to Hemagglutinins from aVariety of Influenza Viruses as Measured by ELISA

The antibodies were tested for binding to hemagglutinins (HAs) fromdifferent influenza strains by ELISA assay. The antibodies were alsoassayed for BSA binding to test for non-specific binding. There is notnecessarily a direct correlation between the binding affinity and thelevel of effectiveness in in vivo studies.

Binding affinity of antibodies was measured by ELISA. To perform theELISA, a 96-well flat bottom NUNC Maxisorp plates (Cat#439454), wascoated with the indicated hemagglutinins (HAs) diluted to 2 μg/mL in1×PBS, 100 μL per well. The plate was sealed with a plate cover, andallowed to incubate overnight at 4° C. static. The plates were thenwashed three times with 1×PBS+0.05% Tween-20 (PBST). The HA-coatedplates were blocked with 200 μL of 5% Blotto (Santa Cruz BiotechnologyCat#sc-2325) in 1×PBS and incubated at room temperature for 1 hour.Subsequently, the plates were washed three times with PBST. Once washed,the antibodies (described in the disclosure) were diluted to the desiredstarting concentration in PBST and loaded on the plate with serialdilution. No sample was added to the last well in the set to get theantigen blank reading. The plates were incubated at room temperature for2 hours, static, and then washed three times with PBST, and tapped todry. Anti-human HRP-conjugated antibody was diluted in PBST, and 100 μLof HRP-antibody was loaded per well to all wells. After incubation for 1hour at room temperature, the plates were washed three times with PBST.TMB solution (KPL, Gaithersburg, Md., Cat#50-76-00) was prepared priorto use and allowed to warm to room temperature. The TMB solution wasthen added to the plate and developed until max OD is between 2 and 3absorbance units at 650 nm as read on a 96-well plate reader (SpectraMaxM2e or similar), or 4 minutes, upon attainment of desired OD, quench thereaction with 1N H₂SO₄ and OD at 450 nm on same plate reader was read onthe plate. The OD was plotted as a function of concentration and therelative Kd was calculated using a four-parametric fit.

Antibody Ab 018.

Antibody Ab 018 was found to have picomolar binding affinity to Group 1strains (H1 (A/Solomon Islands/3/2006), H5 (A/Vietnam/1203/2004) and H9(A/Hong Kong/1073/99)), and at least one Group 2 strain (H7(A/Netherlands/219/2003) but not at least one strain of H3(A/Wyoming/03/2003)) when measured by ELISA. See Table 5.

TABLE 5 Antibody Binding Assays ELISA Kd (pM) Antibody H1 H3 Bris 07 H3Wy03 H5 H7 H9 Ab 018 144 459 VLB 68 845 67 VLB = “Very Low Binding”

Ab 018 also bound with high affinity to H3 from A/Brisbane/10/2007. Thebinding affinity was comparable to H7 HA. Extending the dose rangerevealed low affinity binding of A18 to A/Wyoming/03/2003 (FIG. 5).

Ab 018 also bound with high affinity to both HA₀ and intact virus (FIG.6). The epitope that Ab 018 binds is therefore present on both cleavedand uncleaved HA.

Antibodies Ab 004, Ab 005, Ab 031, Ab 032, Ab 037, and Ab 038.

Binding affinities for select antibodies (Ab 004, Ab 005, Ab 031, Ab032, Ab 037, and Ab 038), as determined by ELISA, are shown in Table 6below.

TABLE 6 Antibody Binding Assays ELISA Kd (pM) Expression H3 H3 H3 H3Rate Antibody H1 (Wyo3) (Bri07) (NY04) (Wis05) H5 H7 H9 (mg/L) Ab 004<80 272 <80 66 713 60 73 60 5.8 Ab 005 <80 311 52 65 1093 38 64 35 4.0Ab 031 <25 331 68 59 253 <25 91 <25 7.4 Ab 032 <25 262 <25 94 208 <25106 79 23.7 Ab 037 75 512 87 140 313 59 114 75 15.1 Ab 038 64 515 <25102 199 87 119 <25 12.0

Antibodies Ab 014 and Ab 028.

Antibodies Ab 014 and Ab 028 were found to have broad specificity, asmeasured by ELISA assay (see FIGS. 10A to 10D).

Antibody Ab 044.

Antibody Ab 044 was found to have less than 500 pM binding affinity (Kd)to hemagglutinins (HAs) from a variety of influenza strains of differentgroups, clades, and subtypes, including Group 1 strains (“the H1acluster” (H1 (A/Solomon Islands/20/1999, A/Puerto Rico/8/34), H2(A/chicken/PA/2004), H5 (A/Vietnam/1203/2004)); “the H9 cluster” (H9(A/Hong Kong/1073/1999, A/Guinea fowl/HK/WF10/99)); “the H1b cluster”(H16 (A/black headed gull/Mongolia/1756/2006))) and Group 2 strains(“the H3 cluster” (H3 (A/Brisbane/10/2007, A/New York/55/2004,A/Wyoming/3/2003, A/Wisconsin/67/2005, A/Moscow/10/1999,A/Perth/16/2006, A/Uruguay/716/2007)); “the H7 cluster” (H7(A/Netherlands/219/2003))), when measured by ELISA.

Antibody Ab 044 was also found to have less than 500 pM binding affinity(Kd) to an HA from influenza B (B/Wisconsin/1/2010) as measured byELISA.

Example 3. Binding of Anti-HA Antibody Ab 032 to Hemagglutinins fromVarious Influenza Strains as Measured by Surface Plasmon Resonance (SPR)

The GE Biacore™ Biotin CAPture Kit (Amersham Biosciences, Pittsburgh,Pa.) has been successfully used to measure the on-rate (k_(on)) andoff-rate (k_(off)), from which the dissociation constant(K_(D)=k_(off)/k_(on)) was obtained between two partners. Specifically,the interaction of Ab 032 against sub-stoichiometrically biotinylatedand immobilized H3 (A/Brisbane/10/07), H7 (A/Netherlands/219/03), H1(A/California/07/09), and H5 (A/Vietnam/1203/2004) hemagglutinins fromProtein Sciences (Meriden, Conn.) were assessed using surface-plasmonresonance on a Biacore® 3000 (Amersham Biosciences, Pittsburgh, Pa.).The kit contains a chip whose surface is covalently modified withsingle-stranded DNA. The kit also contains complementary DNA modifiedwith streptavidin, which is captured by the surface throughhybridization. Biotinylated hemagglutinin targets that have beenpassaged through two Zeba Spin Desalting Columns and Devices 7k MWCO(Thermo Fisher Scientific, Rockford, Ill.) to remove unincorporatedbiotin were applied at the desired RU level (˜100 RU of differenthemagglutinin ligands on flow cells 2, 3, or 4). The biotinylated targetligand was immobilized by the strong, non-covalent biotin-streptavidininteraction, and modification of target lysines with an activated NHSester of biotin (#21338 EZ-Link Sulfo-NHS-LC-LC-Biotin from ThermoFisher Scientific, Rockford, Ill.) was performed sub-stoichiometricallywith respect to the hemagglutinin ˜72 kDa monomer to aid in uniformligand presentation. The kinetic parameters were based on global fittingof 4 or more curves generated by sequential 30 μL/min injections of Ab032 antibody analyte of increasing concentration (500 sec of on-ratedata to fit) and accumulation of the decay data when the injection wasstopped (1200 sec of off-rate data to fit), using double referencesubtraction and a 1:1 binding with mass transfer model with a dockedchip that was normalized with 40% glycerol prior to the experiment.Running buffer and diluent for Ab 032 was 1×PBS (Gibco®, LifeTechnologies Corporation, Grand Island, N.Y.) in 3 mM EDTA (pH 8.5) and0.005% Surfactant P-20 (GE # BR-1000-54, Amersham Biosciences,Pittsburgh, Pa.). The BIAevaluation software (version 4.1.1) was used inthe ‘Kinetics Simultaneous ka/kd . . . ’ module in which the refractiveindex was set to 0, the Rmax was fit locally, and the 1:1 binding withmass transfer was employed. The data were ‘double reference subtracted’before processing, in which the signal from the reference flow cell(containing the streptavidin surface but no hemagglutinin) wassubtracted from the hemagglutinin-containing flow cell of interest; fromthe curves within this set were then subtracted the curve from therunning buffer injection (no antibody). The surface was regenerated byDNA denaturation with an injection of 0.25M NaOH and 6 M guanidine-HClprior to the next cycle of applying fresh DNA-streptavidin andbiotinylated hemagglutinin target for the next Ab 032 concentrationinjection. Three blank injection full run cycles were performed followedby low to high concentrations of Ab 032 analyte, and the third blankinjection was used for double reference subtracting using flow cell 1,which contained hybridized DNA-streptavidin conjugate, but without beingcharged with biotinylated material.

Binding of Ab 032 antibody analyte was measured againstsub-stoichiometrically biotinylated and immobilized hemagglutinin H7Netherlands target. Two-fold serial dilutions of Ab 032 from 8 nM to 0.5nM were used against 85 RU H7 deposited.

In a separate experiment, binding of Ab 032 antibody analyte wasmeasured against sub-stoichiometrically biotinylated and immobilizedhemagglutinin H1 California 07 target. Two-fold serial dilutions of Ab032 from 8 nM to 0.5 nM were used against 111 RU H1 deposited.

The results are shown in Table 7 below.

TABLE 7 Binding Affinity of Ab 032 as Measured by SPR k_(on) k_(on)k_(off) k_(off) K_(D) K_(D) Chi² Antibody:Target (M⁻¹s⁻¹) (error) (s⁻¹)(error) (pM) (error) (error) Ab 032:H3 1.61 × 10⁶ 1.24% 3.76 × 10⁻⁴0.56% 234 1.36% 0.4 (A/Brisbane/10/07) Ab 032:H7 1.13 × 10⁶ 0.34% 1.61 ×10⁻⁴ 0.52% 145 0.62% 0.5 (A/Netherlands/219/03) Ab 032:H1 1.55 × 10⁶0.24% 2.45 × 10⁻⁵ 2.28%  16 2.29% 0.8 (A/California/07/09) Ab 032:H59.88 × 10⁵ 0.23% NA NA NA NA 0.4 (A/Vietnam/1203/2004)

The off-rate (k_(off)) for Ab 032 interacting with H5 Vietnam was soslow, that it fell outside of the range of Biacore measurementspecifications (<5×10⁻⁶ s⁻¹), and is therefore designated as notavailable (NA) in Table 7. Since the dissociation constant (K_(D)) isdetermined by the off-rate divided by the on-rate, it also bears the NAdesignation.

Example 4. In Vitro Antiviral Activity of Anti-HA Antibodies

MIC Assay, CPE Assay, and qRT-PCR.

The antibodies were tested to determine minimum inhibitory concentration(MIC). Despite the results of the ELISA assay (shown in Example 2)indicating that A18 does not bind a strain of H3 (A/Wyoming/03/2003),A18 neutralized strains of both H1N1 (PR8) and a different strain ofH3N2 (X-31) in vitro in a MIC assay. MIC of A18 with PR8 was about 26μg/mL, and MIC of A18 with X-31 was about 421 μg/mL.

The antibodies described in Table 6 also neutralized H1N1 and H3N2viruses in vitro. See below in Table 8.

TABLE 8 Results of MIC Assays MIC (μg/mL) Antibody H1N1 (PR8) H3N2(X-31) Ab 004 12 6 Ab 005 10 10 Ab 031 1 20 Ab 032 3 2 Ab 037 4 8 Ab 0382 4

In a cytopathic effect (CPE) assay, A18 at 10 μg/mL and 50 μg/mLdemonstrated near complete inhibition of X-31 infection while the AB1anti-HA control antibody at 10 μg/mL and 50 μg/mL showed little or noinhibition of X-31 infection (FIG. 4). AB1 binds the stem region of theHA trimer.

A qPCR assay quantification of X-31 viral RNA further indicated that theIC₅₀ of A18 is likely significantly lower than 10 μg/mL.

RT-PCR experiments revealed that A18 could neutralize multiple H3N2strains, including Vic75 (IC₅₀=2 μg/mL), X-31 (IC₅₀=0.4 μg/mL) andBris07 (IC₅₀=˜7 μg/mL).

Visual and Neutral Red Assays.

External validation of in vitro neutralization potential of Ab 032 wasconfirmed by visual and neutral red assays.

Briefly, the antibodies were prepared in MEM solution with 50 μg/mLgentamicin. Starting at 500 μg/mL as the highest concentration, half-logdilutions were prepared and added to 5 wells each on a 96-well platewith MDCK cells in confluency. Three wells of each dilution wereinfected with a low titer of virus, and two wells remained uninfected astoxicity controls. Plates were incubated 3-6 days until virus controlwells reached maximum cytopathic effect (CPE). Plates were eitherevaluated by visual scoring of CPE or stained with neutral red dye forapproximately 2 hours, then supernatant dye was removed from the wellsand the incorporated dye was extracted in 50:50 Sorensen citratebuffer/ethanol, then the optical density (O.D.) was read on aspectrophotometer. O.D. were then converted to percent of cell controlsand normalized to the virus control, then the concentration of testcompound required to inhibit CPE by 50% (EC50) was calculated byregression analysis. The concentration of compound that would cause 50%CPE in the absence of virus was similarly calculated (CC50). Theselectivity index (SI) is the CC50 divided by EC50.

The results are shown below in Table 9.

TABLE 9 Results of Visual and Neutral Red assays for Ab 032 VisualNeutral Red Virus EC₅₀ CC₅₀ SI EC₅₀ CC₅₀ SI^(a) H1N1 Ca04<0.059 >180 >3100 0.2 >180 >900 H1N1 SI <0.059 >180 >31000.08 >180 >2200 H3N2 Bris07 <0.059 >180 >3100 0.076 >180 >2400 H3N2Fujian03 <0.059 >180 >3100 2.2 >180 >82 H3N2 Pan99 19 >180 >9.538 >180 >4.9 H3N2 0.35 >180 >510 1.5 >180 >120 Shangdong93 H3N2 Vic751.1 >180 >160 1.3 >180 >140 H3N2 Wyo03 5.3 >180 >34 >180 >180 0 H5N1Duck81 <0.059 >180 >3100 <0.059 >180 >3100 ^(a)SI is selectivity index(CC50:EC50)

The in vitro antiviral effect of Ab 044 was also examined by the assaydescribed above. The results are shown in Table 10. In vitro, Ab 044demonstrated dose-dependent viral inhibition in vitro with an EC50 inthe range of 0.3-6.8 ug/ml against all Group 1 and Group 2 virus strainstested.

TABLE 10 Results of Neutral Red Assay for Ab 044. Ab 044 (μg/mL) VirusInoc.^(a) EC₅₀ ^(b) CC₅₀ ^(c) SI^(d) H1N1 California 04/2009 3005.1 >500 >98 H1N1 Solomon Islands 160 1.2 >500 >420 H3N2Brisbane/10/2007 40 0.3 >500 >1700 H3N2 Fujian/411/2003 650.3 >500 >1800 H3N2 Panama/2007/99^(e) 13 11 >500 >45 H3N2Shangdong/09/93 100 0.6 >500 >890 H3N2 Victoria/3/75^(e) 124.9 >500 >100 H3N2 Wyoming/03/2003 10 6.8 >500 >74 H5N1 Duck/MN/1525/81160 1.5 >500 >330 ^(a)Inoculum, 50% cell culture infectious dose(CCID50) of virus per well ^(b)CC₅₀ = 50% toxic concentration ofcompound without virus added (μg/mL) ^(c)EC₅₀ = 50% effective antiviralconcentration (μg/mL) ^(d)SI = CC50/EC50 ^(e)Mouse-adapted strain

Example 5. In Vitro Drug Resistance Assay

The emergence of drug resistance was evaluated after continuous exposureof the H1N1 influenza strain PR8 to anti-influenza HA-targetingantibodies described herein. In brief, PR8 was pre-incubated with anantibody at the IC₅₀ for 40 minutes prior to infecting confluent MDCKcells in a 96 well plate format. Infection occurred for 1 h, at whichtime antibody and virus containing media was removed and replaced withvirus-free media containing the antibody at the IC₅₀. After 48 hoursincubation at 37° C., 5% CO₂, the supernatant was removed and viraltiter was quantified by real time PCR using primers specific for thevirus M protein. Once titered, viral supernatants were diluted and usedto re-infect MDCK cells at the same PFU/mL following pre-incubation withdrug at the IC₅₀. As each round of re-infection continues under drugpressure, there is an increased likelihood of selecting for resistantpopulations. C179 was evaluated as a control agent since it is a knownanti-influenza agent that targets the stem region of HA (Okuna et al., JVirol 1993).

An example using two control anti-HA stem antibodies, AB1 and C179(Takara), is shown in FIG. 11. Concentration of both antibodies wasmaintained at 1 μg/mL (starting IC50 levels for both). After 5 rounds ofpassaging in the presence of C179, but not in the presence of AB1, PR8viral titers recovered, suggesting that under given conditions PR8 isresistant to escape from AB1 inhibition but not from that of C179.

Both AB1 and C179 inhibited PR8 propagation in MDCK cells, however thisinhibition was lost over five rounds of passaging with C179 treatmentwhile it was retained during AB1 treatment. Drug passaged PR8 was plaquepurified to isolate ten plaques which were sequenced for HA to confirmthat AB1 was not generating minority resistant populations.

These results indicated that treatment of cells with AB1 preventedproduction of escape mutants for at least five rounds of infectivity.

Example 6. Inhibitions of H5-Mediated Cell Fusion by Anti-HA Antibodies

Ab 032 and Ab 044 were evaluated for their abilities to block cell-cellfusion. Briefly, the assay utilizes HEK293 cells that stably express anddisplay H5 (Viet04). The membrane-anchored hemagglutinin can be inducedto convert to its fusion conformation by a brief (3 minute) exposure tolow pH (5.0). A 3-hour incubation period follows to allow the cells torecover and fuse to form syncytia. A nuclear stain is used to aid in thevisualization of these fusion products, and their count is used as agauge of fusion activity. The test antibody is added either before orafter the low pH treatment to determine with which stage of the fusionprocess it interferes.

Ab 032 or Ab 044 was added at the indicated levels to 96-well platecultures of HEK293 cells displaying surface H5 (Viet04) either 1 hourbefore or immediately after induction of HA into its fusion conformationby a 3 minute low pH (5.0) or neutral pH (7.0; control). After the3-minute induction to stimulate cell fusion, the buffer was replacedwith full culture medium (pH 7.4), and the cells were allowed to recoverand fuse during a 2-3-hour incubation period. Cell cultures were treatedwith Ab 032 at either 0, 10 or 100 μg/mL or with Ab 044 at 0, 0.2, 0.78,3.13, 12.5, or 50 μg/mL, either before or after fusion induction by lowpH or treatment with control buffer at neutral pH. The cells were thenfixed and their nuclei stained with Hema-3. The degree of cell fusionwas measured by the number of syncytia observed under the microscopelens (20×) per field (FIG. 15).

As shown in FIG. 15, Ab 032 is effective in inhibiting cell fusion onlywhen added to the cell culture before the activation of HA by low pH.

As shown in FIG. 18, syncytia formation between HA-expressing cells wasinhibited with pre-treatment of Ab 044 in a dose responsive manner. Theresult indicated that Ab 044 was able to block HA-mediated fusion ifpresent prior to exposure to low pH buffer, presumably by binding to HAand preventing its conversion to the active/fusogenic conformation.Ab044 does not inhibit agglutination of tRBC by H1N1 virus.

Example 7. Prophylactic and Therapeutic Effect of Anti-HA Antibodies ina Murine Co-Infection Model

Therapeutic administration of Ab 032 and Ab 028 rescued mice challengedwith H1N1 and H3N2.

To test the effect of anti-HA antibodies in vivo, a mouse model ofco-infection was used (McCullers, “Effect of Antiviral Treatment on theOutcome of Secondary Bacterial Pneumonia after Influenza” Jour. Infect.Dis. 190:519-526, 2004). Briefly, on day zero, mice were anaesthetizedunder isoflurane and challenged intranasally with influenza H1N1 at 100PFU/head in a volume of 50 μL PBS. On day 7 after viral infection, micewere anesthetized under isoflurane and challenged intranasally withStreptococcus pneumoniae at a dose of 200 CFU/head in a volume of 50 μLPBS. Animals were administered drug by intraperitoneal (IP) treatment ina 200 μL volume on the days as indicated in Table 11. Because theanaesthesia regimen may contribute to the disease state, all mice wereanaesthetized at both infection steps. Lungs were harvested on day fourpost-viral infection for the determination of viral load and on dayeleven post-viral infection for the determination of bacterial load.Lungs were stored at −80° C. until such time as all samples could beanalyzed for viral and microbial loads respectively. Weight and bodyscore of the animals were recorded daily. Animals were euthanized uponloss of considerable weight (>20%) in conjunction with physicalindicators of illness such as piloerection.

A schematic of the experimental design is provided in Table 11 below.The negative control group was co-infected but received no agent otherthan PBS. Ribavirin, a known inhibitor of H1N1, was used as the positivecontrol for antiviral activity. Azithromycin, a known inhibitor ofStreptococcus pneumoniae, was used as to the positive control forantibacterial activity. Ab 032 was administered as prophylaxis in asingle dose 24 h prior to infection at 10 mg/kg or as therapy in asingle dose 48 h after infection at 10 mg/kg. Ab 028 at 10 mg/kg wasadministered as therapy in a single dose 48 h post infection.

TABLE 11 Experimental Design for Murine Co-Infection Model Azithro-Negative Ab 032 Ab 032 Ribavarin mycin Ab 028 Day control (mg/kg)(mg/kg) (mg/kg) (mg/kg) (mg/kg) −1  — 10 — — — — 0 — — — 75 — — (H1N1Challenge) 1 — — — 75 — — 2 — — 10 75 — 10 3 — — — — — — 4 — — — — — —(Lung Harvest) 5 — — — — — — 6 — — — — — — 7 — — — — 10  — (StrepChallenge) 8 — — — — 5 — 9 — — — — 5 — 10  — — — — 5 — 11  — — — — 5 —(Lung Harvest) 12  — — — — 5 — 13  — — — — 5 — 14  — — — — 5 —

Survival curves were generated to describe the experimental outcome.

Ribavirin treatment failed to rescue animals from death while Ab 028therapy resulted in 100% survival (FIG. 16). Ab 032 either asprophylaxis or as therapy at 10 mg/kg protected 80% of mice from deathdue to secondary pneumococcal infection, despite comprising no directantibacterial inhibitory activity (FIGS. 9A, 9B and 9C).

Taken together, Ab 028 and Ab 032 can not only impact viral-induceddamage but also prevent complications from secondary, opportunisticbacterial infections, such as from Streptococcus pneumonia.

Example 8. Prophylactic and Therapeutic Efficacy of Anti-HA AntibodiesAB1 and A18 in H1N1 and H3N2 Mouse Models

The prophylactic and therapeutic efficacy of anti-HA antibodies AB1 andA18 was investigated in the H1N1 and H3N2 mouse models in essentiallythe same way as described in Example 10.

The results are shown in FIGS. 7A-7B. AB1 and A18 were efficacious at 10mg/kg against H1N1 (PR8) when administered to mice 48 h post infection(FIG. 7A).

A18 was also efficacious at 10 mg/kg against H3N2 (Vic75) whenadministered to mice as prophylaxis or 48 h post infection (FIG. 7B).

Example 9. Prophylactic and Therapeutic Efficacy of Anti-HA AntibodiesAb 028, Ab 031, and Ab 032 in H1N1 and H3N2 Mouse Models

The prophylactic and therapeutic efficacy of anti-HA antibodies Ab 028,Ab 031, and Ab 032 was investigated in the H1N1 and H3N2 mouse models inessentially the same way as described in Example 10.

The results are shown in FIGS. 8A-8B. FIG. 8A indicates the survivaltime of

H1N1-infected mice that were administered HA-antibody therapy 48 hoursafter infection. FIG. 8B indicates survival time of H3N2-infected miceadministered HA-antibody therapy 48 hours after infection.

Example 10. Prophylactic and Therapeutic Efficacy of Anti-HA Antibody Ab044 in H1N1 and H3N2 Mouse Models

In vivo experiments were performed to investigate the potential of agentAb 044 as both therapy and prophylaxis in the H1N1 and H3N2 lethal mousemodels. A dose response design was utilized to discriminate the minimumamounts of drug required for therapeutic efficacy. Ab 044 is sometimesreferred to herein as G044, G44, or Ab044,

Briefly, both the H1N1 and H3N2 mouse models were lethal with achallenge dose of PR8 at 100 PFU/head and of Victoria at 10,000PFU/head. Mice were anaesthetized under isoflurane and challenged INwith 50 ul viral suspension. Animals were administered agent IP in avolume of 200 ul, (a) as prophylaxis one day prior to infection, (b) astherapy two days post infection, or (c) as therapy three days postinfection. Weight and appearance of the animals were recorded daily.Animals were euthanized upon loss of considerable weight (>20%) inconjunction with high body score indicating illness. Lungs wereharvested from some animals on day four post infection for thedetermination of viral load by plaque assay. In addition, lungs on dayeight were submitted for histological examination. The study wascompleted as follows (Table 12).

TABLE 12 Experimental Design Dose Time of Influenza Strain Agent (mg/kg)Administration H1N1 PR8 PBS (Vehicle) — 48 h post infection H1N1 PR8Ribavirin (+) 75 3 days treatment H1N1 PR8 Ab 044 10 24 h prior toinfection H1N1 PR8 Ab 044 2.5 24 h prior to infection H1N1 PR8 Ab 0440.6 24 h prior to infection H1N1 PR8 Ab 044 10 48 h post infection H1N1PR8 Ab 044 2.5 48 h post infection H1N1 PR8 Ab 044 0.6 48 h postinfection H1N1 PR8 Ab 044 20 72 h post infection H3N2 Victoria PBS(Vehicle) — 48 h post infection H3N2 Victoria Ribavirin (+) 75 3 daystreatment H3N2 Victoria Ab 044 10 24 h prior to infection H3N2 VictoriaAb 044 10 48 h post infection H3N2 Victoria Ab 044 2.5 48 h postinfection H3N2 Victoria Ab 044 0.6 48 h post infection H3N2 Victoria Ab044 20 72 h post infection

Summary of Results

In lethal influenza challenge models against H1N1 (A/PuertoRico/08/1934; Group 1 virus) or H3N2 (A/Victoria/03/1975; Group 2virus), a single injection of Ab 044 at 10 mg/kg (48 hours postinfection) or 20 mg/kg (72 hours post infection) leads to 100% survivalof mice (n=5 per arm) if administered therapeutically. Survival iscorrelated with secondary metrics, including drop in viral titer andreduction in viral-induced weight loss and body score.

In lethal influenza challenge models against H1N1 (A/PuertoRico/08/1934) or H3N2 (A/Victoria/03/1975), a single injection of Ab 044at up to 10 mg/kg (24 hours pre infection leads to 100% protection ofmice (n=5 per arm) if administered prophylactically. Survival iscorrelated with secondary metrics, including drop in viral titer andreduction in viral-induced weight loss and body score.

The detailed experimental results are presented below.

H1N1 Results

Visual Cues.

Animals were monitored for signs of illness (ruffled fur, hunching)daily. The visual score reflects the average of the group; here, lineswithout point markers reflect the average of recovering survivor(s).

Mice that were challenged with H1N1 appeared sick three days postinfection and were euthanized on day seven, as expected. Mice that werechallenged with

H1N1 and treated with ribavirin exhibited negligible sign of illness andrecovered fully. Mice that were treated with Ab 044 one day prior tochallenge at 2.5 mg/kg or 10 mg/kg exhibited no sign of illness. Micethat were treated with Ab 044 one day prior to challenge at 0.6 mg/kgexhibited signs of illness; 60% recovered.

Agent Ab 044 was administered in a dose response manner two days postinfection. Animals that received 10 mg/kg exhibited little sign ofillness while animals that received 2.5 mg/kg or 0.6 mg/kg became quiteill with some deaths counted in both groups. Agent Ab 044 was alsoadministered at 20 mg/kg three days post infection; those animalsbenefited from therapy, with a clear difference between treated anduntreated animals on day six, and full recovery by day seven.

Animals were also monitored for weight loss. The weight change reflectsthe average of the group; here, lines without point markers reflect theaverage of recovering survivor(s).

Mice that were challenged with H1N1 lost >20% weight by day seven andwere euthanized, as expected. Mice that were challenged but treated withribavirin at 75 mg/kg once per day for three days exhibited <10% weightloss and recovered. Mice that were treated with Ab 044 one day prior tochallenge at 2.5 mg/kg or 10 mg/kg exhibited no weight loss or gainedweight over time. Mice that were treated with Ab 044 prophylaxis at 0.6mg/kg lost a substantial amount of weight, with three to of five animalslosing ≥16% body weight; two of five animals were euthanized (FIG. 19A).

Ab 044 therapy was administered in a dose response manner Animals thatreceived 10 mg/kg exhibited 10% weight loss and recovered. Animals thatreceived 2.5 mg/kg lost substantial weight with four of five animalslosing >16% body weight; three animals were euthanized. All but oneanimal that received 0.6 mg/kg lost >20% body weight and were euthanized(FIG. 19B).

In summary, Ab 044 prophylaxis one day prior to challenge preventeddeath from H1N1 infection when administered at ≥2.5 mg/kg. Ab 044therapy two days post infection rescued mice from death whenadministered at 10 mg/kg while Ab 044 therapy three days post infectionat 20 mg/kg was fully efficacious.

Viral Load.

The lung viral loads four days after H1N1 infection were assessed in asingle plaque assay (Table 13). The reductions in lung viral load weremore substantial than expected in three groups: ribavirin, Ab 044prophylaxis at 10 mg/kg, and Ab 044 prophylaxis at 2.5 mg/kg. The viralloads in the samples were confirmed by a repeat plaque assay then againby qPCR (data not shown) and thus reported here.

TABLE 13 Lung Viral Load in Mice Four Days after Challenge with H1N1 PR8Dose Lung Viral Load Log Reduction Treatment Group (mg/kg) H1N1 (PFU/ml)from Untreated Untreated — 6.03 — Ribavirin 75 4.38 1.65 Ab 044prophylaxis 10 4.45 1.58 Ab 044 prophylaxis 2.5 4.08 1.95 Ab 044prophylaxis 0.6 5.38 0.65 Ab 044 therapy at 48 h 10 5.34 0.69 Ab 044therapy at 48 h 2.5 5.49 0.54 Ab 044 therapy at 48 h 0.6 5.74 0.29 Ab044 therapy at 72 h 20 5.29 0.74

Comparisons were made between treatment groups to assess thesignificance of the reductions in lung viral load. Significance (p<0.05)was determined Mann Whitney U test. The lung viral load in all treatmentarms was significantly different from that in the untreated group, withthe exception of the Ab 044 therapy at 0.6 mg/kg at 48 h which was nodifferent from untreated.

H3N2 Results

Visual cues.

Animals were monitored for signs of illness (piloerection, hunching)daily.

Mice that were challenged with H3N2 appeared sick three days postinfection and were euthanized on day seven, as expected. Mice that werechallenged with H3N2 and treated with ribavirin exhibited no sign ofillness and recovered fully, as expected. Mice that were treated with Ab044 one day prior to challenge at 10 mg/kg exhibited no sign of illness.

Agent Ab 044 was administered in a dose response manner two days postinfection. Animals that received therapy at ≥2.5 mg/kg exhibitednegligible illness and recovered fully. Mice that received therapy 0.6mg/kg were indistinguishable from mice in the untreated group, withsevere illness and euthanasia required by day seven. Agent Ab 044 wasalso administered at 20 mg/kg three days post infection; those animalsbenefited from therapy, with a clear difference between treated anduntreated animals on day four, and full recovery by day six.

Animals were also monitored for weight loss (FIGS. 20A-20B).

All mice that were challenged with H3N2 lost >10% weight by day five andwere euthanized with >20% weight loss on day seven. Mice that werechallenged but treated with ribavirin at 75 mg/kg once per day for threedays exhibited <10% weight loss and recovered. Mice that were treatedwith Ab 044 one day prior to challenge at 10 mg/kg gained weight overtime (FIG. 20A).

Ab 044 therapy was administered in a dose response manner Animals thatreceived 10 mg/kg exhibited <10% weight loss and recovered. Animals thatreceived 2.5 mg/kg lost >10% weight but recovered. Animals that received0.6 mg/kg lost >20% body weight and were euthanized (FIG. 20B).

Viral Load.

The lung viral loads four days after H3N2 infection were assessed in asingle plaque assay (Table 15).

TABLE 15 Lung Viral Load in Mice Four Days after Challenge with H3N2Dose Lung Viral Load Log Reduction Treatment Group (mg/kg) H3N2 (PFU/ml)from Untreated Untreated — 6.48 — Ribavirin 75 5.66 0.82 Ab 044prophylaxis 10 5.42 1.06 Ab 044 therapy at 48 h 10 5.46 1.02 Ab 044therapy at 48 h 2.5 5.99 0.49 Ab 044 therapy at 48 h 0.6 6.44 0.04 Ab044 therapy at 72 h 20 5.64 0.84

Comparisons were made between treatment groups to assess thesignificance of the reductions in lung viral load. Significance (p<0.05)was determined Mann Whitney U test. The lung viral load in all treatmentarms was significantly different from that in the untreated group, withtwo exceptions. The lung viral loads after Ab 044 therapy at 48 h at 2.5mg/kg or 0.6 mg/kg were no different from untreated.

Correlation Between In Vitro and In Vivo Activities.

Agent Ab 044 exhibited reproducible in vitro activity against H1N1 PR8and H3N2 X31 (Table 17).

TABLE 17 In vitro activity of Agent Ab 044 as Measured by CPE H1N1 H3N2Assay # NB Ref Agent Lot # PR8 X31 27 56-33 Ab 044 202165 13 13 27 56-33Ab 044 202175 12 12 27 56-33 Ab 044 202176 18 9 27 56-33 Ab 044 20217714 14 28 56-56 Ab 044 202188 8 14

In vitro activity has translated into in vivo activity in both models.The lung viral load on day four provided a snapshot of the infectionstate (FIGS. 21A-21B) and demonstrated a significant (p<0.05) reductionin viral load attributable to various treatment strategies with Ab 044.

A second snapshot of the infection was taken when lungs were harvestedfor histological examination on day eight (Tables 14 and 16).Administration of Ab 044 at 10 mg/kg as prophylaxis one day prior tochallenge substantially decreased the severity of necrosis andinflammation attributable to H1N1 infection. Therapeutic administrationof Ab 044 at 10 mg/kg two days after infection had no clear effect onthe disruption of fine lung structure attributable to H1N1 or H3N2infections. The expectation was that both Ab 044 prophylaxis and therapywould visibly reduce the inflammation and necrosis associated withinfluenza infection. Instead, there appears to be a timing component,such that delivery of Ab 044 in advance of the infection alters thecytokine cascade with its recruitment of white cells and resultinginflammation and necrosis while delivery of Ab 044 after infectionminimally impacts the outcome.

Survival Curves Generated for the H1N1 Model.

Ab 044 prophylaxis resulted in 100% survival despite lethal challengewhen administered at >2.5 mg/kg one day prior to infection (FIG. 22A).100% survival of lethally infected animals was observed with Ab 044therapy two days post infection at 10 mg/kg or Ab 044 therapy three dayspost infection at 20 mg/kg (FIG. 22B).

Survival Curves Generated for the H3N2 Model.

Ab 044 prophylaxis at 10 mg/kg resulted in 100% survival (FIG. 23A).100% survival of lethally infected animals was observed with Ab 044therapy two days post infection at ≥2.5 mg/kg or Ab 044 therapy threedays post infection at 20 mg/kg (FIG. 23B).

In summary, Ab 044 was efficacious in both the H1N1 and H3N2 mousemodels (Table 18). Administration of Ab 044 prophylaxis at 2.5 mg/kg orhigher resulted in 100% survival in the H1N1 model, a dose that wouldlikely also achieve to 100% survival in the H3N2 model. Administrationof Ab 044 therapy at 48 h post infection at 10 mg/kg against the H1N1infection and at ≥2.5 mg/kg against the H3N2 infection achieved 100%survival. Administration of Ab 044 therapy at 20 mg/kg three days postinfection rescued 100% of H1N1- and H3N2-infected animals.

TABLE 18 In vivo Efficacy of Agent Ab 044 in Lethal Mouse ModelsSurvival (%) Dose H1N1 H3N2 Agent Administration (mg/kg) (PR8) (Vic75)Untreated — 0 0 0 Ribavirin Days 0, 1, 2 75 100 100 Ab 044 24 h prior toinfection 10 100 100 2.5 100 nd 0.6 60 nd Ab 044 48 h post infection 10100 100 2.5 40 100 0.6 20 0 Ab 044 72 h post infection 20 100 100

Example 11. Prophylactic and Therapeutic Efficacy of Anti-HA Antibody Ab044 in a Highly Pathogenic Avian Influenza A Virus Mouse Model

Ab 044 was tested to determine its efficacy in a highly pathogenic avianinfluenza A H5N1 mouse model.

The objective of this study was to evaluate both prophylactic andtherapeutic dosing regimens of Ab 044 for efficacy. Theparameters/endpoints to be assessed included: weight loss (assessedevery day for 21), virus lung titer reduction at day 4 post virusexposure, and mortality.

Materials and Methods

Animals: Female 17-20 g BALB/c mice were obtained from Charles RiverLaboratories (Wilmington, Mass.) for this study. They were maintained onWayne Lab Blox and tap water ad libitum. They were quarantined for 24 hprior to use.

Viruses: Influenza A/Vietnam/1203/2004 (H5N1) virus was obtained fromDr. Jackie Katz of Centers for disease control. Mice exposed to lethaldose of the virus (5 MLD50, 5 PFU/mouse) generally die from days 8-13.

Experimental design: Groups of mice were intraperitoneally (i.p.)administered Ab 044 one time (qd) 24 h before virus exposure with dosesof Ab 044 at 20, 10, or 5 mg/kg. Five of those mice were sacrificed onday 4 to determine the extent of observable gross lung pathology, theextent of lung edema in the form of increased lung weights, and todetermine virus lung titers. The remaining mice were observed formortality or for adverse events until day 21 following virus exposure.Thirteen mice were treated with 20 mg/kg Ab 044 24 h after virusexposure and 13 mice were treated with 20 mg/kg Ab 044 48 h after virusexposure with 4 mice from each group being sacrificed on day 4 postvirus exposure as described of above, the remaining kept for observationof death or adverse events until day 21 following exposure to virus. Anadditional 7 mice received oseltamivir at 30 mg/kg/d, twice a day forfive days (bid×5) beginning at time 0; being observed for mortality oradverse events until day 21 after virus exposure. Twenty mice were alsotreated with PSS 48 h after virus exposure, and 5 of these weresacrificed for lung titers on day 4. These mice represented the placebocontrols. Mice were weighed every day up to day 21, or until death,beginning just prior to virus exposure.

Lung virus titer determination: Each mouse lung was homogenized in MEMsolution and assayed in triplicate for infectious virus in MDCK cells.Samples from each test group were titered in triplicate.

Statistical analysis: Normality of the animal weights was assessed byD'Agostino & Pearson omnibus normality test. Upon finding that theweight data fit a Gaussian distribution, statistical inferences weremade by two way analysis of variance followed by pairwise comparisonsusing Bonferroni's post-tests to compare each treatment group to theplacebo treatment. Survival analysis was done using the Kaplan-Meiergraphing method and a Logrank test. That analysis revealed significantdifferences among the treatment groups. Therefore, pairwise comparisonsof survivor curves (PSS vs. any treatment) were analyzed by theGehan-Breslow-Wilcoxon test to determine which treatment group differedsignificantly from the placebo group, and the relative significance wasadjusted to a Bonferroni-corrected significance threshold for the numberof treatment comparisons done.

Hazard ratios (HR), which compare how rapidly groups of treated mice aredying relative to untreated control groups of mice, were determined bythe Mantel-Haenszel tests as part of the survival analysis program usedabove (GraphPad Prism® for MAC v5). The Kruskal-Wallis test, followed byDunn's posttest for evaluating significant pairwise comparisons, wasused to detect significant differences in mean day of death betweentreatment groups and the placebo-treated mice. Differences in the ratiosof live mice/total mice for treatment groups were analyzed usingcontingency table analysis, and pairwise comparisons to theplacebo-treated group were made by Fisher's exact tests.

Virus lung titers from each treatment group were compared to untreatedcontrols using the analysis of variance on log-transformed valuesassuming equal variance and normal distribution. When significance atP<0.05 was achieved by ANOVA analysis of all treatments, individualtreatment values were then compared to the PSS control usingNewman-Keuls pair-wise comparison tests.

Summary of Results

In a lethal influenza challenge model against H5N1 (A/Vietnam/1203/2004;Group 1 virus), a single injection of Ab 044 at 10-20 mg/kg (24 hourspre-infection) or 20 mg/kg (up to 72 hours post infection) leads to 100%survival of mice (n=5 per arm) if administered therapeutically. Survivalis correlated with secondary metrics, including drop in viral titer andreduction in viral-induced weight loss and body score. In this sameexperiment, 30 mg/kg of oseltamivir, administered twice daily for 5days, resulted in only 60% survival.

Mice treated with Ab 044 at 10 mg/kg were observed for 14 days. Ab044-treated mice did not lose weight or show any visible signs ofstress. Histological analysis was performed on lungs harvested on day 8,where tissues were scored on the basis of observed necrosis or signs ofinflammation. Neither necrosis nor inflammation was observed in thetissues of mice treated with Ab 044.

The detailed experimental results are presented below.

Results

The weight data shown in FIG. 24 suggest that the compound was welltolerated even in infected mice, since mice treated with Ab 044 did notlose significant amounts of weight compared to the starting weights ofthese treated mice. In addition, the Ab 044-treated, infected mice weresignificantly protected against weight loss due to virus infection atall concentrations of Ab 044 used from days 7 to 12 post virus exposureregardless of the dosing regimen used (FIG. 24, Table 19;P<0.01-P<0.001). The only exception to this generalization was the groupof mice that was treated with Ab044 at 5 mg/kg/d (qd×1, beg. −24 h) onday 12 post virus exposure. Especially notable about the lack of weightloss was the fact that mice treated with the 20 mg/kg doses did not loseweight at all and most mice even had gained weight compared to theirstarting weight by the end of the experiment. This correlated very wellwith the fact that mice receiving Ab 044 at 20 mg/kg post virus exposureall survived the infection (FIG. 25). In fact, significant numbers(90-100%) of mice survived the infection in each treatment groupreceiving Ab 044 regardless of dose and treatment regimen used(P<0.0001).

TABLE 19 Table of Significant Weight Differences Illustrated in FIG. 24.Level of Significance Treatment vs. PSS Day 7 Day 8 Day 9 Day 10 Day 11Day 12 Day 13 Day 14 Ab 044 (20 mg/kg/d, qd X 1, −24 h) P < 0.001 P <0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 Ab 044(10 mg/kg/d, qd X 1, −24 h) P < 0.01 P < 0.001 P < 0.001 P < 0.001 P <0.001 P < 0.01 P < 0.001 P < 0.001 Ab 044 (5 mg/kg/d, qd X 1, −24 h) P <0.01 P < 0.01 P < 0.001 P < 0.001 P < 0.001 NS^(a) P < 0.001 P < 0.001Ab 044 (20 mg/kg/d, qd X 1, +24 h) P < 0.001 P < 0.001 P < 0.001 P <0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 Ab 044 (20 mg/kg/d, qd X1, +48 h) P < 0.01 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P < 0.001 P <0.001 P < 0.001 Oseltamivir (30 mg/kg/d, bid X 5, 0 h) P < 0.01 P <0.001 NS NS P < 0.05 NS P < 0.01 P < 0.001 ^(a)Not Significant (P >0.05).

Although only 60% of mice treated with oseltamivir survived theinfection, this survival rate was significantly different from theinfected untreated group of mice (P=0.0055). In the past, whenH5N1-infected mice have been treated with oseltamivir at 30 mg/kg/d foreight days instead of the five days as was done in the current study,90-100% of mice treated for the longer period of time survived the virusinfection with little or no weight loss. The weight loss in past studieswas significantly less than for untreated, infected mice, unlike theweight loss detected at days 9, 10, and 12 post virus exposure in thecurrent study (FIG. 24, Table 19).

Interestingly, it was during the time period of days 9-12 post virusexposure that mice in the oseltamivir-treated group of mice succumbed tothe virus infection in the current study (FIG. 25).

Not only did treatment of mice with Ab 044 significantly protect miceagainst death as measured by total survivors (Table 20, Live/Totalcolumn, P<0.0019), but the treatments also profoundly affected thekinetics of death; mice treated with Ab 044 were 11-38 times less likelyto die as rapidly from virus infection, if at all, than untreated,infected mice (Table 20, hazards ratios). In addition, for most Ab 044treatment regimens the one mouse that did succumb to infection in eachgroup did so one to two days later compared to mice that died in theplacebo (PSS) group (Table 20, see mean day of death).

TABLE 20 Effects of Ab 044 on Various Mortality Parameters Measured forBALB/c Mice Infected with a Lethal Dose of Influenza A/Vietnam/1203/2004H5N1 Virus Animals: Female 17-20 g BALB/c Mice Treatment Schedule:Variable Virus: Ia/Vietnam/1203/2004 Treatments: (H5N1) 1 LD90 Ab 044Virus route: i.n. Oseltamivir Duration of Experiment: 21 days Treatmentroute: i.p. Mean Day Median Live/ of Death ± Survival Hazard RatioTreatment Total SD^(a) (days)^(b) (95% CI)^(c) PSS 0/15 11.3 ± 1.8 11.5— Ab 044 9/10** 10.0 ± 0.0 Undefined 10.9 (3.9-32.3) (20 mg/kg/d, qd X1, −24 h) Ab 044 9/10** 12.0 ± 0.0 Undefined 14.2 (4.7-42.6) (10mg/kg/d, qd X 1, −24 h) Ab 044 9/10** 13.0 ± 0.0 Undefined 15.74(5.3-46.7)  (5 mg/kg/d, qd X 1, −24 h) Ab 044 9/9^(††) >21*** Undefined 38.0 (12.2-116.6) (20 mg/kg/d, qd X 1, +24 h) Ab 044 9/9^(††) >21***Undefined  38.0 (12.2-116.6) (20 mg/kg/d, qd X 1, +48 h) Oseltamivir4/7* 11.3 ± 0.6 Undefined  4.8 (1.6-14.3) (30 mg/kg/d, bid X 5, 0 h)^(a)The average time to death among animals succumbing to the infection.^(b)The time at which fractional survival equals 50%. (i.e., thecalculated time at which half the subjects have died and half are stillalive.) ^(c)Hazard ratios are calculated relative to the placebo.Live/Total: *P = 0.02, **P = 0.0019 vs. Placebo, ^(††)P = 0.0014 vs.Placebo. Mean Day of Death: ***P < 0.001 vs. Placebo.

No gross lung pathology or edema was detected at day 4 post virusexposure, because these infection related phenomena are usually observedat day 8 or later after virus exposure in the H5N1 mouse model. However,the virus lung titers were significantly for reduced in mice treateddoses of Ab 044 at 10 mg/kg or greater regardless of when Ab 044 wasadministered (Table 21, P<0.05, P<0.01). The virus lung titers from micetreated with 5 mg/kg of Ab 044 were lower, but statistically similar tothe virus lung titers detected in the lungs of mice from the placebogroup.

Thus, reduction of virus lung titers early in the infection by Ab 044treatment at 24 h post virus exposure likely reduced the amount ofantigen produced that was capable of inducing a pathogenichyperinflammatory response characteristic of H5N1 pulmonary infectionsin mice (Otte et al., Am J Pathol 179:230-239 (2011)). However, thevirus titer data from the treatment beginning at 48 h post virusexposure seemingly could contradict that hypothesis, since day 4 titersfor that group of mice were near the levels of the placebo-treated mice.It may be that treatment at 48 h kept the titers sufficiently low forthe next 24 h to avoid a hyper-inflammatory response to the originalinsult. It is also possible that the compound did not have sufficienttime to exert its antiviral effect between 48 hrs and the 4-day virustiter assay, but it had sufficient activity by day 9 when deaths beganto protect the mice from death.

TABLE 21 Effects of Ab 044 on Lung Virus Titer Measured for BALB/c MiceInfected with a Lethal Dose of Influenza A/Vietnam/1203/2004 H5N1 VirusAnimals: Female 17-20 g BALB/c Mice Treatment Schedule: Variable Virus:Ia/Vietnam/1203/2004 Treatments: (H5N1) 1 LD90 Ab044 Virus route: i.n.Oseltamivir Duration of Experiment: 21 days Treatment route: i.p.Treatment Lung Virus Titers ± SD PSS 5.67 ± 0.49 Ab 044 (20 mg/kg/d, qdX 1, −24 h)  3.54 ± 0.91** Ab 044 (10 mg/kg/d, qd X 1, −24 h)  3.01 ±1.05** Ab 044 (5 mg/kg/d, qd X 1, −24 h) 4.40 ± 0.62 Ab 044 (20 mg/kg/d,qd X 1, +24 h)  3.98 ± 0.98* Ab 044 (20 mg/kg/d, qd X 1, +48 h) 5.24 ±0.88 *P < 0.05, **P < 0.01 vs. Placebo.

In conclusion, at doses of 2.5 and 10 mg/kg, Ab 044 gives 100%protection from lethal challenge of H1N1 and H3N2, respectively.Furthermore, when the monoclonal antibody was administered up to 72hours after infection, it was completely effective in treating theinfection with 100% survival for both H1N1 and H3N2 virus subtypes. Ab044, regardless of the dose or dosing regimen used (prophylactic ortherapeutic), was extremely efficacious in protecting BALB/c miceagainst death due to an extremely lethal infection with a highlypathogenic avian influenza A H5N1 virus. Moreover, the same Ab 044treatment regimens were extremely effective in preventing weight lossdue to virus infection and in significantly reducing day 4 virus lungtiters.

Example 12. Competition Assays for Antibody Epitope Mapping

Competition ELISA is used to test whether a monoclonal antibody (mAb1)alters the ability of another antibody (e.g., mAb2, or test antibody) tobind the target. To perform the assay, a 96-well flat bottom NUNCMaxisorp plates (Cat#439454) is coated with the desired hemagglutinin(HA) diluted to 2 μg/mL in 1×PBS, 100 μL per well. The plate is sealedwith a plate cover, and allowed to incubate overnight at 4° C. static.The plates are then washed thrice with 1×PBS+0.05% Tween-20 (PBST). TheHA-coated plates are blocked with 200 μL of 5% Blotto (Santa CruzBiotechnology Cat#sc-2325) in 1×PBS and incubated at room temperaturefor 1 hour. Subsequently, the plates are washed thrice with PBST. Onehundred microliters of mAb1 is then added to the wells at saturationconcentration (determined previously) and incubated for 2 hours at roomtemperature. Post-incubation, the plates are washed to remove unboundmAb1 and the test antibody (mAb2) is diluted to the desired startingconcentration in PBST and loaded on the plate. The plates are incubatedat room temperature for 2 hours, static and then washed thrice withPBST, and tapped to dry. Appropriate HRP-conjugated antibody at desiredconcentration is diluted in PBST, and 100 μL of HRP-antibody is loadedper well to all wells. After incubation for 1 hour at room temperature,the plates are washed thrice with PBST. TMB solution (KPL Cat#50-76-00)is prepared prior to use and allowed to warm to room temperature. TheTMB solution in then added to the plate and developed until max OD isbetween 2 and 3 absorbance units at 650 nm as read on a 96-well platereader (SpectraMax M2e or similar). Upon attainment of desired OD, thereaction quenched with 1N H₂SO₄ and the OD at 450 nm is read. The OD isplotted as a function of concentration and the Kd is calculated using afour-parametric fit. The existence of an overlapping epitope betweenmAb1 and mAb2 will be indicated by a significant drop in binding of mAb2to HA in the presence of mAb1 relative to that observed in its (mAb1)absence in control wells.

Example 13: Making and Testing of Candidate Antibodies

Recombinant Expression of Antibody:

For recombinant expression of an IgG1, the V_(H) and V_(L) regions ofthe antibody can be either isolated from B-cells, hybridomas orsynthesized and sub-cloned into plasmids containing CH1-H2-H3 and CLrespectively. Recombinant expression of antibody can be carried out inmammalian cells such as HEK 293-F FreeStyle suspension cells(Invitrogen, Carlsbad, Calif.) cultured in 293-F FreeStyle ExpressionMedium (Invitrogen, Carlsbad, Calif.) maintained at 37° C., 80% humidityand 8% CO₂. Cells (with >95% viability) are transfected withPoly-ethylene-imine Max (PEI-MAX, PolySciences) with equivalent amountsof HC and LC containing plasmids. Seven days post-infection, the cellsare harvested by spinning the cells at 4000 rpm for 15 min at 4° C. andfiltered through a 0.45 μm filter system (Nalgene) and supplemented with1:1000 diluted protease inhibitor cocktail (Calbiochem).

The antibody is purified from the supernatant using a Protein A resin(Pierce) filled column on a AKTA Purifier FPLC system. The antibody iseluted with a 100 mM Glycine-HCl buffer (pH 2.5) buffer and pHneutralized by the addition of 10% 1M Tris-base 2.5 M NaCl (pH 8.5). Thepurified sample is then buffer exchanged into 1×PBS (pH 7.4) andconcentrated by ultrafiltration/diafiltration (UF/DF) using a 30 KDaMWCO spin filter (Millipore). Purified antibody is quantified usingNanoDrop spectrophotometer.

ELISA Binding Assays

Recombinant hemagglutinin is diluted to 2 μg/mL in PBS and 100 μl isused to coat 96-well microtiter plates (Immuno™ Maxisorp, Nunc). Theplates are incubated at 4° C. overnight. The plates are subsequentlywashed thrice with 1×PBS+0.05% Tween-20 (PBST) and then blocked with 200μL of 5% Blotto (Santa Cruz Biotechnology) in 1×PBS for 1 hr. The platesare then thrice washed with PBST and antibody serially diluted in PBSTis added and incubated for 2 hrs at room temperature. The wells aresubsequently washed with PBST and the bound IgG1 is detected using 100μl of 1:1000 HRP-conjugated anti-hIgG1. After 1 hr incubation, theplates are washed and the reaction was developed using TMB solution(KPL) and stopped by addition of 1N H₂SO₄. The absorbance at 450 nm ismeasured using d TMB on a SpectraMax M2e plate reader.

Microneutralisation Assays

In vitro neutralization assay is performed using the protocol describedby Sidwell and Huffman to test the ability of the antibody to inhibitinfectivity of influenza virus in MDCK cells. Briefly, the antibody isprepared in half-log dilutions starting with 500 μg/mL in MEM solutionwith 50 μg/mL of gentamicin. Each dilution is added to 5 wells of a96-well plate with confluent cells. Three wells of each dilution isinfected with a low titer of virus, and two wells remain uninfected astoxicity controls. Ribavirin is included as a control. Plates areincubated 3-6 days until virus control wells reached maximum cytopathiceffect (CPE). Plates are then stained with neutral red dye forapproximately 2 hours, then supernatant dye is removed from the wellsand the incorporated dye is extracted in 50:50 Sorensen citratebuffer/ethanol, and the optical density is read on a spectrophotometer.Optical densities are converted to percent of cell controls andnormalized to the virus control, and the concentration of test compoundrequired to inhibit CPE by 50% (EC₅₀) is calculated by regressionanalysis. The concentration of compound that would cause 50% CPE in theabsence of virus was similarly calculated (CC₅₀). The selective index(SI) is the CC₅₀ divided by EC₅₀.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims

We claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence that encodes a heavy chain immunoglobulin variable region or alight chain immunoglobulin variable region of antibody moleculecomprising: (a) a heavy chain immunoglobulin variable region segmentcomprising: a CDR1 comprising the sequence S-Y-A-M-H (SEQ ID NO:68); aCDR2 comprising the sequence V-V-S-Y-D-G-N-Y-K-Y-Y-A-D-S-V-Q-G (SEQ IDNO:69); and a CDR3 comprising the sequenceD-S-R-L-R-S-L-L-Y-F-E-W-L-S-Q-G-Y-F-N-P (SEQ ID NO:70); and (b) a lightchain immunoglobulin variable region segment comprising: a CDR1comprising the sequence Q-S-I-T-F-D-Y-K-N-Y-L-A (SEQ ID NO:145); a CDR2comprising the sequence W G S Y L E S (SEQ ID NO:72); and a CDR3comprising the sequence Q Q H Y R T P P-S(SEQ ID NO:73).
 2. Arecombinant vector comprising the nucleic acid molecule of claim
 1. 3. Acell comprising the vector of claim
 2. 4. A method of making an antibodymolecule, comprising providing a cell of claim 3 and expressing thenucleic acids in the cell, thereby making the antibody molecule.
 5. Thenucleic acid molecule of claim 1, wherein the antibody moleculecomprises a heavy chain immunoglobulin variable region segment thatcomprises SEQ ID NO:
 25. 6. The nucleic acid molecule of claim 1,wherein the antibody molecule comprises a light chain immunoglobulinvariable region segment that comprises SEQ ID NO:
 52. 7. The nucleicacid molecule of claim 1, wherein the antibody molecule comprises aheavy chain immunoglobulin variable region segment that comprises SEQ IDNO: 25 and a light chain immunoglobulin variable region segment thatcomprises SEQ ID NO:
 52. 8. The nucleic acid molecule of claim 1,wherein the antibody molecule comprises a tetramer of two heavy chainimmunoglobulin variable region segments, each comprising SEQ ID NO: 25,and two light chain immunoglobulin variable region segments, eachcomprising SEQ ID NO:
 52. 9. The nucleic acid molecule of claim 1,wherein the antibody molecule comprises a full length antibody.
 10. Thenucleic acid molecule of claim 1, wherein the antibody moleculecomprises a humanized antibody molecule.
 11. The nucleic acid moleculeof claim 1, wherein the antibody molecule comprises two heavy claimvariable regions and two light chain variable regions.
 12. The nucleicacid molecule of claim 1, wherein the antibody molecule is an IgGantibody.
 13. The nucleic acid molecule of claim 1, comprising anucleotide sequence that encodes a heavy chain immunoglobulin variableregion and a light chain immunoglobulin variable region of the antibodymolecule.
 14. The nucleic acid molecule of claim 1, comprising the heavychain variable region nucleotide sequence of SEQ ID NO: 151, the lightchain variable region nucleotide sequence of SEQ ID NO: 149, or both.15. A recombinant vector comprising the nucleic acid molecule of claim7.
 16. A recombinant vector comprising the nucleic acid molecule ofclaim
 14. 17. A cell comprising the vector of claim
 15. 18. A cellcomprising the vector of claim
 16. 19. A method of making an antibodymolecule, comprising providing a cell of claim 17 and expressing thenucleic acid molecules in the cell, thereby making the antibodymolecule.
 20. A method of making an antibody molecule, comprisingproviding a cell of claim 18 and expressing the nucleic acid moleculesin the cell, thereby making the antibody molecule.